CN1314706C - Antigen epitope II of neutrality B cell of chicken IBDV VP2 protein and application thereof - Google Patents
Antigen epitope II of neutrality B cell of chicken IBDV VP2 protein and application thereof Download PDFInfo
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- CN1314706C CN1314706C CNB2005100174536A CN200510017453A CN1314706C CN 1314706 C CN1314706 C CN 1314706C CN B2005100174536 A CNB2005100174536 A CN B2005100174536A CN 200510017453 A CN200510017453 A CN 200510017453A CN 1314706 C CN1314706 C CN 1314706C
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Abstract
The present invention belongs to the field of molecular immunology, which mainly relates to series polypeptide fragment fragments of chicken infectious bursa disease virus (IBDV) VP2 proteins, particularly to series polypeptide fragments for forming B cell antigen epitope in VP2 proteins. The present invention simultaneously relates to the application of IBDV VP2 protein neutrality B cell antigen epitope for preparing immunogen, immunizing animals, generating immune protection and detecting antibodies or polypeptide antibodies for resisting chicken IBDV. After immunogen or vaccine designed by chicken IBDV VP2 protein neutrality B cell antigen epitope immunizes animal organisms, neutrality antibodies aiming at IBDV can be generated, IBDV in vivo or in vitro can be neutralized, virus is prevented from infecting animal organisms, and the epitope vaccine of chicken IBDV VP2 protein neutrality B cell antigen epitope can prevent the infection of chicken IBD classic strains, virulent strains or/and variant strains.
Description
One. technical field: the present invention relates to the field of immunology of molecule, particularly relate to a series of chicken infectivity bursa of Fabricius virus IBDV VP2 albumen neutrality B cell antigen epi-positions and application thereof.
Two. background technology:
Infectious bursal disease (Infectious bursal disease, IBD) be the chicken that causes by chicken infectivity bursa of Fabricius virus (IBDV) acute, height contact, kill the transmissible disease of lymphatic.Nineteen fifty-seven takes place in the fryer group in the Gumboro town in the Delaware state of the U.S. first, claims the Gan Buluo disease again.The Winterfield isolation identification went out IBDV in 1962.This disease all has generation and popular in the U.S. and countries in the world.Caused enormous economic loss in China's big area outbreak of epidemic in 1988 ~ 1992 years.Discover that IBD is a kind of immunologic injury disease based on humoral immune reaction.IBDV belongs to birnavirus section (Birnaviridae), Avibirnavirus (avianbirnavirus), and this virus has two serotypes, and the serum I type causes a disease to chicken, and the II type only infects turkey but is not pathogenic.The IBDV virus particle is the individual layer capsid, and no cyst membrane is the icosahedron symmetry, and diameter is 60-70nm, and buoyant density is 1.31-1.34 in the cesium chloride, and is insensitive to EC, highly antiacid.
Chicken infectivity bursa of Fabricius virus IBDV is a diplornavirus, and its genome is by A, and two fragments of B are formed.The A fragment is made up of 3200-3300bp Nucleotide, and the B fragment is made up of 2800-2900 Nucleotide.The A fragment contains a big open reading frame (ORF) and a little ORF.The virus precursor polyprotein (polyprotein) that big ORF coding is made up of 1012 amino acid, molecular weight is about 110kD.This precursor protein is by VP2, and VP4 and VP3 form.The VP5 albumen of the about 17kD of little ORF coding molecule amount.B fragment encoding viral protein VP1, this albumen are the dependent RNA polymerase of RNA, with virus duplicate and assemble relevant.
VP2 albumen is the main protection antigen of IBDV, contains neutrality B cell antigen epi-position.Protective antigen is the recommendation antigen that uses as vaccine.The B cell antigen epi-position that has neutralizing effect in protective antigen plays an important role in stimulating the neutrality antibody generation.Epitope claims antigenic determinant again, and being meant can be by antibody, TCR/MHC mixture in conjunction with the antigene fragment of also discerning.Wherein, can be called the B cell antigen epi-position by antibodies and identified epitope; Can be called the T cell antigen epitope by combination of TCR/MHC mixture and identified epitope.After virus antigen entered body, the antigen receptor molecule sIgM on protective antigen and bone-marrow-derived lymphocyte surface and IgD combined and cause bone-marrow-derived lymphocyte activation and cloning increment, and produce the antibody at the specific b cells epitope, bring into play immanoprotection action.Therefore, the B cell antigen epi-position is determining body to produce the specificity of antibody, is the fundamental unit of inducing humoral immunization.Concerning the proteantigen of specific virus, identify that its B cell antigen epi-position is to disclose the essence of viral humoral immunization.
Method commonly used during proteantigen B cell antigen epi-position is identified has genetically engineered rite-directed mutagenesis antigen expressed analysis of protein method, random phage peptide storehouse technology and synthetic polypeptide detection technique etc.Rite-directed mutagenesis antigen expressed protein process is one of the most frequently used technology, this method is convenient, simple, cost is low, but, this method can only identify the antigenic key amino acid of performance site in the proteantigen roughly, can not finally determine the short amino acid sequence of this epitope, can not distinguish this epitope and belong to linear epitope or structure as the property epi-position.The phage peptide library technology can analog representation and ligands specific bonded peptide sequence, can go out and a certain antigen specific antibodies bonded epitope by sequencing analysis through behind the multi-turns screen, not only linear epitope can be measured, and structure can also be simulated as the property epitope.Synthetic polypeptide technology can identify linear B cell epitope specific in a certain proteantigen the most clearly.This invention using gene engineering technique overlapping (overlapping) is expressed the VP2 structural protein of IBDV, with among the tentatively definite VP2 of external immune response with specificity neutrality monoclonal antibody bonded zone; Then, use the synthetic polypeptide technology of peptide storehouse technology and overlapping and determine that short amino acid sequence of linearity on these structural protein and simulation structure are as epi-position; At last, use the exactness of synthetic polypeptide technical identification epi-position again.
Three. summary of the invention:
The objective of the invention is: a series of chicken infectivity bursa of Fabricius virus IBDV VP2 albumen neutrality B cell antigen epi-positions and their application are provided.
Technical scheme of the present invention is:
A kind of chicken infectivity bursa of Fabricius virus IBDV VP2 albumen neutrality B cell antigen epi-position, its aminoacid sequence is Seq No 1:
197H-Cys-Asp-Ser-Ser-Asp-Arg-Pro-Arg-Val-Tyr-Thr-Ile-Thr-OH209。
Described chicken infectivity bursa of Fabricius virus IBDV VP2 albumen neutrality B cell antigen epi-position, according to chicken infectivity bursa of Fabricius virus IBDV VP2 Argine Monohydrochloride sequence extension, the aminoacid sequence after the expansion is Seq No 2 to its aminoacid sequence to the C end:
197H-Cys-Asp-Ser-Ser-Asp-Arg-Pro-Arg-Val-Tyr-Thr-Ile-Thr-Ala-Ala-Asp-Asp-Tyr-Gln-Phe-Ser-Ser-Gln-Tyr-Gln-Pro-Gly-Gly-OH224。
A kind of chicken infectivity bursa of Fabricius virus IBDV VP2 albumen neutrality B cell antigen epi-position, its aminoacid sequence is Seq No 3:
327H-Trp-Ser-Ala-Arg-Gly-Ser-Leu-Ala-Val-Thr-Ile-His-Gly-OH339。
Described chicken infectivity bursa of Fabricius virus IBDV VP2 albumen neutrality B cell antigen epi-position, according to chicken infectivity bursa of Fabricius virus IBDV VP2 Argine Monohydrochloride sequence extension, the aminoacid sequence after the expansion is Seq No 4 to its aminoacid sequence to the C end:
327H-Trp-Ser-Ala-Arg-Gly-Ser-Leu-Ala-Val-Thr-Ile-His-Gly-Gly-Asn-Tyr-Pro-Gly-Ala-Leu-Arg-Pro-Val-Thr-Leu-Val-OH352。
Described chicken infectivity bursa of Fabricius virus IBDV VP2 albumen neutrality B cell antigen epi-position is synthetic polypeptide.
Described chicken infectivity bursa of Fabricius virus IBDV VP2 albumen neutrality B cell antigen epi-position is carried out chemically modified at N end or C end.
Described chemically modified is the natural amination or the acetylize of polypeptide chain N end, or the carboxylated naturally or amidation of C end.
Described chicken infectivity bursa of Fabricius virus IBDV VP2 albumen neutrality B cell antigen epi-position or its connector are in the preparation immunogen, or immune animal, or produce the application on the immunoprotection.
Described chicken infectivity bursa of Fabricius virus IBDV VP2 albumen neutrality B cell antigen epi-position or its connector are in the application that detects on anti-chicken infectivity bursa of Fabricius virus IBDV antibody or the anti-chicken infectivity bursa of Fabricius virus IBDV polypeptide antibody.
Immunogen or the vaccine formed by epitope polypeptide that the present invention relates to, at least comprise that an epitope of mentioning in the claim is to whole epitopes, these polypeptide connect, interconnect or are connected with carrier by self, and be aided with the T cell antigen epitope, comprise Th1 and/or Th2 epitope polypeptide.These T cell antigen epitopes can derive from has the immunocompetent peptide sequence of the body cell of stimulation in IBDV viral protein or other animal proteinum.
Detect chicken IBDV virus and can use at the monoclonal antibody of VP2 or use at the VP2 polypeptide antibody, detection method can comprise that agar diffusion test, indirect ELISA, double-antibody sandwich elisa, quick detection test paper bar immunity rete analyse technology etc.
Detect chicken IBDV antibody, comprise that maternal antibody, vaccine immunity antibody, wild virus infection produce antibody, anti-chicken IBDV monoclonal antibody etc.Detection method can comprise that agar diffusion test, indirect ELISA, double-antibody sandwich elisa, quick detection test paper bar immunity rete analyse technology etc.
Positive beneficial effect of the present invention is:
1. by the neutrality antibody that can produce behind the immunogen of chicken infectivity bursa of Fabricius virus IBDV VP2 albumen neutrality B cell antigen epi-position design or the vaccine immunity animal body at IBDV, and can be in vivo or in external and IBDV, stop the virus infection animal body.
2. immunogen or the vaccine by chicken infectivity bursa of Fabricius virus VP 2 albumen neutrality B cell antigen epi-position design of the present invention can prevent chicken IBD classical strains, highly virulent strain or/and the infection of variation strain.
3. chicken infectivity bursa of Fabricius virus VP 2 albumen neutrality B cell antigen epi-position of the present invention is connected with carrier or self connects or interconnects; can immune animal, anti-peptide antibody that when immune animal, produces or anti-chicken infectivity bursa of Fabricius virus antibody can be in vivo and in vitro in and chicken infectivity bursa of Fabricius virus IBDV virus and produce immunoprotection.
4. anti-peptide antibody that chicken infectivity bursa of Fabricius virus VP 2 albumen neutrality B cell antigen epi-position of the present invention or its connector produce when immune animal or anti-IBDV antibody can detect IBDV virus or its polypeptide.
5. chicken infectivity bursa of Fabricius virus VP 2 albumen neutrality B cell antigen epi-position of the present invention or its connector can detect anti-chicken infectivity bursa of Fabricius virus IBDV antibody or anti-chicken infectivity bursa of Fabricius virus IBDV polypeptide antibody.
Four. description of drawings:
Fig. 1 is epitope polypeptide Seq No 1 and Seq No 3 detection chicken IBDV serum antibody and monoclonal antibody results: be followed successively by IBDV positive serum 1, positive serum 2, IBDV monoclonal antibody, BSA contrast, negative serum contrast among the figure from left to right.
Five. embodiment:
Embodiment one: the pulsating gene clone of chicken infectivity bursa of Fabricius virus IBDV VP2 albumen neutrality B cell antigen epi-position:
SPF kind egg is hatched to 11 ages in days, gets Embryo Gallus domesticus and prepares chick embryo fibroblast, and inoculation IBDV-H4 strain virus was cultivated 96 hours, and collected virus for 37 ℃.With ultracentrifugation and 40 ~ 60% sucrose density gradient centrifugation isolated virals, obtain virus particle.With TRIZOL method extracting virus total RNA, handle geneome RNA with Proteinase K, the degradation of rna duplex molecule is as template applications RT-PCR method amplification IBDV A segment genome.Reclaim purified pcr product, and be connected with the pGEM-T carrier, transformed into escherichia coli makes up A segment genome clone.
Be used for the serial primer that overlapping expresses viral protein, pcr amplification VP2 and VP2 gene segment according to VP2 constitutional features and antigenicity analysis consequence devised.Make up VP2 and pulsating prokaryotic expression carrier thereof, and at pET system prokaryotic expression virus antigen, the virus antigen of expression prepares immunogen after the dissolving renaturation, immune balb/c mice, preparation monoclonal antibody.
Carry out the series screening of IBDV neutrality monoclonal antibody according to the phage peptide library handbook of Biolabs company.1. with going forward side by side property of LB/IPTG/xgal amplification M13 phage peptide library titer determination; 2. the affine magnetic bead of IBDV monoclonal antibody labelled immune; 3. immune affine magnetic bead and 2 * 10
11The phage reaction 60min of amplification; 4. PBST washes 10 times, the unconjugated phage of flush away; 5. using immune affine magnetic separation technique collects and IBDV monoclonal antibody bonded phage; 6. wash-out and monoclonal antibody bonded phage; 7. the amplification of wash-out bacteriophage and titer determination; 8. repeat 3. ~ 7. the step operation carry out the peptide storehouse second to the three-wheel elutriation; 9. select the locus coeruleus bacterium colony from the LB/IPTG/xgal flat board after the third round elutriation and increase, extract phage DNA and carry out sequencing; 10. further identify the reactivity of phage-displayed polypeptides with IBDV quick detection test paper bar.Use the Characterization of antigenic epitopes programanalysis of peptide storehouse at last and determine that clone's material is a chicken infectivity bursa of Fabricius virus IBDV VP2 albumen neutrality B cell antigen epi-position.
Embodiment two: Fmoc solid-phase polypeptide synthesis method overlapping synthesizing series VP2 antigen peptide segment is also carried out the Dot-ELISA screening:
According to the indirect ELISA monoclonal antibody response spectrum of prokaryotic expression VP2 and overlapping expression VP2 antigene fragment, and the VP2 neutralizing antibody site sequence of phage peptide library technology screening, determine that overlapping synthesizes the first step sequence of polypeptide.And use characteristics such as the Chou and Fasman algorithm second structure characteristic of these polypeptide fragments of Computer Analysis, wetting ability, antigenicity, surperficial accessibility, the sequence of polypeptide is synthesized in design.And use the synthetic difficulty characteristic of peptide programanalysis, design polypeptide synthesis program is used Peptide synthesizer and is carried out the synthetic automatically and manual synthetic method synthetic peptide sequence that combines.Idiographic flow is as follows:
Design synthetic peptide sequence → peptide programanalysis → design synthesis program → calculate and take by weighing Fmoc-AA-Wang-resin or Rink resin → DMF swelling resin → * by the substitution value of resin adds 20%piperidine and takes off Fmoc and protect; stirring 6min → * adds next bit amino acid and HBTU carries out acylation reaction, N
2Stirring reaction 30min → * washes 5 times with the performance → * DMF of Kaiser method or TNBS method test reaction, each 1min → repeat to have step of *, on resin, connect next bit amino acid, up to this peptide sequence is synthetic finishes → choose reactivity → analysis IBDV neutrality monoclonal antibody reactivity B cell antigen epi-position that suitable reagent is tested neutrality monoclonal antibody and polypeptide with coupling → Dot-ELISA of being connected of TFA method cracking peptide chain and resin → cold diethyl ether precipitation TFA polypeptide → Sephadex G-25 desalting and purifying → LC-MS and HPLC Analysis and Identification and purified polypeptide → polypeptide and carrier according to component peptide chain amino acid difference.
Embodiment three: chicken infectivity bursa of Fabricius virus IBDV VP2 albumen neutrality B cell antigen epi-position synthetic:
According to the aminoacid sequence of VP2 albumen neutrality B cell antigen epi-position, use the solid-phase polypeptide synthetic technology, adopt automatic Peptide synthesizer or artificial polypeptide synthesis method.Solid-phase resin adopts amino acid Wang resin or other resin of Fmoc protection, and resin according to the aminoacid sequence order, adds the amino acid of Fmoc protection after DMF swelling, piperidine take off the Fmoc protection, exist at HBTU and carry out acylation reaction under the situation.Through washing, add deputy Fmoc-amino acid again and carry out acylation reaction after acylation reaction is finished, and washing.So circulation is initial to N-terminal from the peptide sequence C-terminal, according to the synthetic complete polypeptide chain of sequence order.After synthetic the finishing, choose suitable reagent with being connected and precipitating the TFA polypeptide of TFA method cracking peptide chain and resin with cold diethyl ether according to component peptide chain amino acid difference, standby after desalting and purifying, LC-MS and HPLC Analysis and Identification.In entire synthesis process, can use the performance of Kaiser method or TNBS method test reaction after each amino acid acylation reaction.For the ease of epitope polypeptide is connected with carrier proteins, when synthesizing polypeptide, can add a halfcystine at the N-terminal of peptide sequence.
With aminoacid sequence 81H-Lys-Phe-Asp-Gln-Met-Leu-OH86 is that synthetic 5 required reagent of μ mol polypeptide of example and operating process are as follows:
Main agents: N, N-dimethylformamide (DMF); Deprotecting regent I:20%Piperidine/DMF; Deprotecting regent II: methylene dichloride (DCM); Condensation, activating reagent: 2-(1H-benzo three)-N, N, N ', a N ' tetramethyl-urea hexafluorophosphate (HBTU) is dissolved in NMM (N-methylmorpholine)/DMF; Cutting reagent: 94% trifluoroacetic acid (TFA); 20 kinds of Fmoc protection amino acid (25 μ mol/L): Fmoc-Ala-OH; Fmoc-Cys (Trt)-OH; Fmoc-Asp (OtBu)-OH; Fmoc-Glu (OtBu)-OH; Fmoc-Phe-OH; Fmoc-Gly-OH; Fmoc-His (Trt)-OH; Fmoc-Ile-OH; Fmoc-Lys (Boc)-OH; Fmoc-Leu-OH; Fmoc-Met-OH; Fmoc-Asn (tBu)-OH; Fmoc-Pro-OH; Fmoc-Gln (Trt)-OH; Fmoc-Arg (Pbf)-OH; Fmoc-Ser (tBu)-OH; Fmoc-Thr (tBu)-OH; Fmoc-Val-OH; Fmoc-Trp (Boc)-OH; Fmoc-Tyr (tBu)-OH.Main resin: Fmoc-Leu-Wang-Resin; Symphony Peptide synthesizer: U.S. Protein Technology company.
Amino acid coupling step during polypeptide is synthetic: according to purpose peptide chain output, weighing 10mg Fmoc-Leu-WangResin puts into reaction flask; (1) adds 1.25ml DMF, mixed 10 minutes, blow DMF off, repeat more than 3 times with N2; (2) add 1.25ml 20%Piperidine/DMF, mixed 5 minutes, blow liquid off, add 1.25ml 20%Piperidine/DMF, hybrid reaction 15 minutes with N2; (3) repeating step (1); (4) Fmoc-Met-OH of adding 1.25ml 25 μ M; Add 1.25ml 0.4NMM/DMF, hybrid reaction blew liquid off with N2 more than 45 minutes; (5) repeating step (1); (6) repeating step 1～5 adds Fmoc-Met-OH successively, Fmoc-Gln (Trt)-OH, and Fmoc-Asp (OtBu)-OH, Fmoc-Phe-OH, Fmoc-Lys (Boc)-OH finish each amino acid whose coupling in the peptide sequence; (7) nitrogen dries up resin.
The cutting basic step of the synthetic purpose peptide that finishes is as follows: (1) adds 1.25ml DMF, mixes 10 minutes, blows DMF off with N2, repeats more than 3 times; (2) add 1.25ml 20%Piperidine/DMF, mixed 5 minutes, blow liquid off, add 1.25ml 20%Piperidine/DMF, mixed 15 minutes with N2; (3) repeating step (1); (4) wash more than three times with DCM; (5) add the 94%TFA hybrid reaction more than 2 hours; (6) collect cleaved products.
Polypeptide synthesis product is slightly carried: anhydrous diethyl ether slowly added in the product of collection, and ice bath 10 minutes, 3000rpm, centrifugal 10 minutes, stay precipitation, abandon supernatant, repeat 3 times, throw out Freeze Drying Equipment drying ,-20 ℃ of preservations are standby.The sample that takes a morsel is used for HPLC, and GC-MS detects.
Embodiment four: the chemically modified of chicken infectivity bursa of Fabricius virus IBDV VP2 albumen neutrality B cell antigen epi-position:
According to the method synthetic polypeptide fragments of embodiment three, its N-terminal is that nature is amido modified, and C-terminal is the nature carboxyl modified.
N-terminal acetylation modification method: the polypeptide synthesis method according to above introduction is blended into last Fmoc protection amino acid coupling end, and carries out the removal of N-terminal Fmoc protection group, carries out following operation then.Get 150 μ l (2.6 μ mol) diacetyl oxide and 20 μ l (0.1 μ mol) EIPEA is dissolved among the 4.8mlDMF, thorough mixing also cools off in ice bath.Then above mixing solutions is added in the polypeptide building-up reactions bottle and reacted 5 minutes.Use 5ml DMF and methylene dichloride (DCM) to wash again successively 2 times, each 5 minutes.Nitrogen dries up the back and carries out removal, cracking, purifying and the evaluation of side chain protected group according to aforementioned conventional synthetic method.
C-terminal amidation modifying method: should select resin for use when the amidated polypeptide of C-terminal is synthetic is the Rink resin.The reply resin is used DMF swelling 20 minutes when carrying out synthesis program, begins to carry out synthesis program from C-terminal first amino acids then, with aforementioned Fmoc-wang resin synthetic method.
Embodiment five: chicken infectivity bursa of Fabricius virus IBDV VP2 albumen neutrality B cell antigen epi-position is connected with carrier proteins KLH and BSA's:
Experiment material comprises: epitope polypeptide; Hemocyanin (KLH), Pierce company; Bovine serum albumin (BSA), DMSO, Sigma company; Linking agent Sulfo-SMCC, Pierce company product.
Carrier proteins KLH or BSA 4mg are dissolved in the PBS damping fluid that 0.5ml contains 5mM EDTA, and pH 7.2, add 1.0mg linking agent Sulfo-SMCC, and fully hatched 30 minutes in incubated at room in 60 minutes or 37 ℃ the dissolving back.Use the carrier proteins that Sephadex G-25 desalting column or dialysis method purifying Sulfo-SMCC handle, and measure protein content with BCA protein determination kit (Pierce company), standby.
Epitope polypeptide is synthetic finish after, accurately weighing 4mg adds 0.5ml bi-distilled water dissolving polypeptide, can to add an amount of DMSO short molten if the polypeptide solvability is bad.Polypeptide after will dissolving then mixes with the carrier proteins that Sulfo-SMCC handles, and hatches 2 hours or spends the night for 4 ℃, and is standby.
Embodiment six: the oxidation of chicken infectivity bursa of Fabricius virus IBDV VP2 albumen neutrality B cell antigen epi-position realizes the connection of self:
The epitope polypeptide that contains halfcystine can form self and connect by the oxidizing reaction of sulfydryl.The oxidizing reaction of using the DMSO mediation generally speaking realizes connecting, and according to the acid-basicity of polypeptide, oxidizing reaction can be carried out under little acid or slight alkalinity situation.
Oxidizing reaction under the slightly acidic environment: with the acetic acid water dissolution polypeptide of proper concn, the concentration that makes polypeptide is in the 0.5-1.5Mm scope, the final concentration of acetic acid is no more than 5%, the pH that adjusts polypeptide solution with (NH4) 2CO3 is about 6.0, the DMSO that adds 10-20%, 25 ℃ of reaction 5-25h, the carrying out degree and can monitor of oxidizing reaction by HPLC.Then, be moving phase with 1%TFA water and acetonitrile, preparation type reversed-phase HPLC purifying oxidisability polypeptide, standby.
Oxidizing reaction under the slight alkalinity environment: use the 0.01M phosphate buffered saline buffer, pH7.5, the dissolving polypeptide adds the DMSO of final concentration 1% to final concentration 1.0mM, and 25 ℃ of reactions are spent the night, the carrying out degree and can monitor by HPLC of oxidizing reaction.Then, be moving phase with 1%TFA water and acetonitrile, preparation type reversed-phase HPLC purifying oxidisability polypeptide, standby.
Embodiment seven: chicken infectivity bursa of Fabricius virus IBDV VP2 albumen neutrality B cell antigen epi-position is used the connection of linking agent:
The epitope polypeptide that contains the epitope polypeptide of halfcystine and do not contain halfcystine is undertaken self and connects, interconnects or be connected with carrier by amino glutaraldehyde or carbodiimide (EDC) the homotype bifunctional reagents such as (Fluka company products) used of N end α amino or Methionin ε.
Take by weighing 1mg BSA with activating damping fluid (0.1M MES, 0.5M NaCl, pH5.7) be made into the solution that concentration is 1mg/ml, add 0.4mg EDC (2mM) and 0.6mg NHS (Fluka product), room temperature reaction is 15 minutes behind the thorough mixing, adding final concentration is the mercaptoethanol of 20mM, adds the 1.0mg epitope polypeptide, room temperature reaction 2 hours; Then, add the 0.7mg oxammonium hydrochloride, room temperature reaction 30 minutes; At last, using Sephadex G-25 desalting and purifying, is the balance elute soln with the activation damping fluid, and the 280nm ultraviolet wavelength detects, and collecting first, to flow out component be the polypeptide connector, and 4 ℃ or-20 ℃ of preservations are standby after sterile filtration.
Embodiment eight: chicken infectivity bursa of Fabricius virus IBDV VP2 albumen neutrality B cell antigen epi-position indirect ELISA detects the test of chicken IBDV antibody:
The connector of VP2 albumen neutrality B cell antigen epi-position or epitope polypeptide and carrier is an envelope antigen, is contrast antigen with BSA, and bag is by 96 hole enzyme plates (polystyrene micropore plate).Use pH9.6,0.1M carbonate buffer solution dilution envelope antigen adds in the enzyme plate 4 ℃ of overnight incubation to final concentration 1.0 μ g/ml by 50 μ l/ holes.Use PBST washing lotion (PBS+0.05%Tween) detersive enzyme target 6 times then, 2%BSA washing lotion sealase target 2 hours is used to detect IBDV antibody.
When detecting antibody, go into operation down successively: add serum to be checked (or carry out doubling dilution serum be used to detect antibody titer), hatched 30-60 minute PBST washing lotion detersive enzyme target 6 times for 37 ℃; Add goat-anti chicken IgG horseradish peroxidase ELIAS secondary antibody (Goat-antichicken IgG-HRP) or anti-chicken IgG monoclonal antibody-HRP ELIAS secondary antibody, hatched 30-60 minute PBST washing lotion detersive enzyme target 6 times for 37 ℃; Add HRP substrate and developer, the room temperature lucifuge is hatched and was observed color reaction in 5-10 minute; After treating fully colour developing, add 2% sulfuric acid and stop color reaction; Read the light absorption value of reaction plate hole at wavelength 450nm with microplate reader (Bio-Rad); Result of determination.
During result of determination, when serving as 3 times of contrast plate hole light absorption values, be judged to reacting positive with the light absorption value of sample plate hole; Maximum dilution multiple with reacting positive serum is the antibody titer of this sample serum.
Embodiment nine: chicken infectivity bursa of Fabricius virus IBDV VP2 albumen neutrality B cell antigen epi-position Dot-ELISA detects the test of chicken IBDV antibody:
The connector of VP2 albumen neutrality B cell antigen epi-position or epitope polypeptide and carrier is an envelope antigen, is contrast antigen with BSA.Go up quantitative specking envelope antigen with a film instrument (Bio-Dot) at nitrocellulose filter (Millipore), 1-2 μ g/ point is fixed 30 minutes for 37 ℃, PBST washing 6 times, and the sealing of 10% skimming milk washing lotion is spent the night, and PBST washing 6 times promptly can be used for antibody test.
When detecting antibody, with serum sample to be checked quantitatively dilute the back and antigen coated film hatched 30 minutes for 37 ℃, PBST washing 6 times, add goat-anti chicken IgG horseradish peroxidase ELIAS secondary antibody (Goat-antichickenIgG-HRP) or anti-chicken IgG monoclonal antibody-HRP ELIAS secondary antibody, hatched 30-60 minute PBST washing lotion detersive enzyme target 6 times for 37 ℃; Add the colour developing of AEC or DAB substrate, the room temperature lucifuge is hatched and was observed color reaction in 5-10 minute.
When the result judges, present the red-brown color reaction with the envelope antigen point, contrast antigen point color reaction do not occur and judges.
Embodiment ten: neutrality immunogenic preparation of B cell antigen epi-position of chicken infectivity bursa of Fabricius virus IBDV VP2 albumen and immunization experiment:
The antigen of chicken IBDV VP2 albumen neutrality B cell antigen epitope antigen during as immunogen preparing.Head exempts from antigen with Freund's complete adjuvant (Sigma) and antigen emulsification, with every polypeptide 5-10 μ g immune animal; Two, three use Freund's incomplete adjuvant (Sigma) and antigen emulsification when exempting from, with every polypeptide 5-10 μ g immune animal.Immunization route is subcutaneous or intramuscular injection.Immunity time: head exempts from the age in days into 7-14, and two exempted from back 14 days headed by exempting from, and can carry out booster immunization according to the immune antibody level later on.
Embodiment 11: use the example that the VP2 monoclonal antibody detects chicken infectivity bursa of Fabricius virus:
Use VP2 monoclonal antibody detection chicken IBDV virus and can adopt agar diffusion test, the double-antibody sandwich elisa test, and bispecific monoclonal antibody immunity rete is analysed test strip test etc.Tissues such as the desirable chicken bursa of sample to be checked, liver, spleen, kidney, with these tissues in 1: 5 ratio with physiological saline or PBS damping fluid homogenized, centrifugal 30 minutes of 3000rpm, it is to be checked to get the supernatant suspension.
When agar diffusion test detects, make a call to 7 hole plum blossom holes in agar plate, interstitial hole adds the VP2 monoclonal antibody, peripheral hole adds sample to be checked, standard positive sample and negative sample etc., and each 50 μ l of every hole put into wet box, hatched observations 12-24 hour for 37 ℃.The result judges precipitation line to occur between standard positive sample and the VP2 monoclonal anti body opening, when precipitation line not occurring between negative sample and the VP2 monoclonal anti body opening, the positive sample of sample that occurs precipitation line between sample well and the VP2 monoclonal anti body opening, otherwise negative sample.
When the double-antibody sandwich elisa test detects, be the coated antibody coated elisa plate with a strain VP2 monoclonal antibody, bag is 10 μ g/ml by concentration, and method for coating is undertaken by embodiment eight.During detection, the sample adding to be checked of suitably dilution is wrapped by good enzyme plate, hatched 30 minutes for 37 ℃ in 50 μ l/ holes, PBST washs 6 times, adds another strain VP2 monoclonal antibody of HRP mark then, and hatched 30 minutes for 37 ℃ in 50 μ l/ holes, PBST washing 6 times is with the colour developing of HRP substrate, result of determination.
When bispecific monoclonal antibody immunity rete is analysed the test strip detection, with a strain VP2 monoclonal antibody and Radioactive colloidal gold coupling spraying glass wool, another strain VP2 monoclonal antibody and rabbit anti-mouse igg are sprayed at respectively on the nitrocellulose filter as detection line and control line, according to the assemble method of test strip sample pad, glue gold cotton, nitrocellulose filter, absorbent pad being assembled into immune rete by the assembling mode of test strip then analyses test strip (detailed method is with reference to Chinese invention patent ZL99101537.1, " livestock and poultry pestilence quick detection test paper bar " Zhang Gaiping etc., 1999), be used for detecting.During detection, the test lead of test strip is inserted 10-20 second in the sample liquid to be checked of suitably dilution, kept flat after the taking-up 1-5 minute, then result of determination.Test strip is judged to the positive when two lines (detection line and control line) occurring, promptly this chicken infects IBDV virus, and test strip is judged to feminine gender when a control line only occurring.
Embodiment 12: the example of using anti-VP2 epitope polypeptide antibody test chicken infectivity bursa of Fabricius virus:
The preparation of chicken infectivity bursa of Fabricius virus IBDV VP2 albumen neutrality B cell antigen epitope antibodies is with embodiment ten, and sample preparation and detection method are with reference to embodiment 11 when detecting chicken IBDV virus.
Embodiment 13: use chicken infectivity bursa of Fabricius virus IBDV VP2 albumen neutrality B cell antigen epi-position and detect chicken IBDV antibody:
Detect synthetic polypeptide and anti-chicken IBDV monoclonal antibody or the sero-fast reactivity of chicken IBDV with Peptide-Dot-ELISA.Method by embodiment five after epitope polypeptide Seq No 1 and Seq No 3 balanced mix is connected the preparation envelope antigen with carrier proteins BSA.Go up quantitative specking BSA-Pep with a film instrument (Bio-Dot) at nitrocellulose filter (Millipore), 37 ℃ of fixing 30min, PBST washing 6 times; 10% skimming milk PBST seals nitrocellulose filter, overnight incubation, PBST washing 6 times; The envelope antigen film at reactive tank and example reaction to be checked (anti-chicken IBDV monoclonal antibody or chicken IBDV antiserum(antisera)), is hatched 30-60min for 37 ℃, PBST washing 6 times; Add the GAM-IgG-HRP or the RACh-IgY-HRP working solution of PBST dilution in 1: 100 in reactive tank, hatch 30-60min for 37 ℃, PBST washes 6 times; Adding substrate solution AEC, RT is hatched 10min, after treating fully to develop the color, cleans the color development stopping reaction with PBST, and observations is the positive sample that reddish-brown develops the color, and the result is as shown in Figure 1.
Embodiment 14: the immunity test of chicken infectivity bursa of Fabricius virus IBDV VP2 albumen neutrality B cell antigen epi-position:
Method by embodiment five after epitope polypeptide Seq No 1 and Seq No 3 balanced mix is connected with carrier proteins KLH, the preparation polypeptide immunogen.5 Balb/c mouse of immunity, control group are used irrelevant rondom polypeptide immunity.Immunizing dose 50 μ g/, first adjuvant-free uses FCA, and two, three times immunological adjuvant uses FIA.Carry out immunity by 0,14,28 age in days immune programme for children, 42 ages in days blood sampling separation of serum is measured antibody titer and is carried out neutralization test with Peptide-ELISA.Use fixed virus dilute serum method and carry out the neutralization test of polypeptide antibody.Test is carried out in 96 well culture plates, and every hole contains 10
4Chick embryo fibroblast (CEF).Mouse anti polypeptide (Seq No 1 and Seq No 3) antibody, IBDV monoclonal antibody (positive control), behind irrelevant immediately polypeptide antibody (negative control) doubling dilution of mouse anti, every hole adds 10
2TCID
50IBDV.Behind the room temperature neutralization reaction 45min, adding contains in the CEF cell hole, in 37 ℃, and 5%CO
2Cultivate observation of cell pathology (CPE) in the incubator, and calculate neutralization and tire.
Use indirect ELISA and detect the mouse anti polypeptide antibody, the result shows that 4 (M1, M2, M4 and M5) in 5 immune peptide mouse produced the anti-peptide antibody (table 1) of high titre, and it is tired and can reach 1: 3200 to 1: 6400.Neutralization test shows that anti-peptide antibody has significant neutralization activity to serum I C-type virus C (H4 highly virulent strain and D78 low virulent strain), and anti-irrelevant polypeptide antibody does not then show neutralization active (table 2)
Table 1 mouse anti epitope polypeptide antibody indirect ELISA is tired
| Mouse number | ELISA titres a | ||||||
| 1∶200 | 1∶400 | 1∶800 | 1∶1600 | 1∶3200 | 1∶6400 | Blank | |
| M1 M2 M3 M4 M5 M-control b | 1.543 1.863 0.366 1.366 1.004 0.205 | 1.248 1.490 0.212 0.991 0.902 0.167 | 0.984 1.050 0.143 0.660 0.945 0.108 | 0.821 0.697 0.108 0.401 0.711 0.091 | 0.634 0.439 0.091 0.233 0.567 0.085 | 0.525 0.290 0.085 0.153 0.279 0.077 | 0.090 0.087 0.078 0.072 0.077 0.076 |
aEnvelope antigen is epitope polypeptide AG-3 and the LG-4 that BSA connects.
b5 mean values that the irrelevant polypeptide mice serum of immunity is tired.
Table 2 anti-peptide antibody is to the neutralization activity of serum I type IBDV
a
| The IBDV strain | Neutralization is tired b | ||||
| The anti-IBDV antibody of chicken | Mouse-anti epitope polypeptide antibody c | Monoclonal antibody | Monoclonal antibody | The mouse-anti polypeptide antibody that has nothing to do d | |
| H4 B87 | 16.5 17.0 | 15.6 14.5 | 17.0 16.5 | 18.0 17.5 | ＜3.5 ＜3.5 |
aFixed virus on the CEF cell (100TCID50)/dilution antibody neutralization test
bThe logarithm of the highly diluted multiple of serum that occurs with no CPE or monoclonal antibody is tired for the neutralization of this antibody
cMouse anti epitope polypeptide antibody is M1, M2, the mixing of M4 and M5 mice serum
dThe serum that the irrelevant polypeptide antibody of mouse anti is 5 control mice mixes
Sequence table
＜110〉Henan Science and Technology College
＜120〉chicken IBDV VP2 albumen neutrality B cell antigen epi-position II
<130>seq002
<160>4
<170>PatentIn version 3.1
<210>1
<211>13
<212>PRT
＜213〉Avibirnavirus (Avibirnavirus gen.)
<400>1
Cys Asp Ser Ser Asp Arg Pro Arg Val Tyr Thr Ile Thr
1 5 10
<210>2
<211>13
<212>PRT
＜213〉Avibirnavirus (Avibirnavirus gen.)
<400>2
Trp Ser Ala Arg Gly Ser Leu Ala Val Thr Ile His Gly
1 5 10
<210>3
<211>28
<212>PRT
＜213〉Avibirnavirus (Avibirnavirus gen.)
<400>3
Cys Asp Ser Ser Asp Arg Pro Arg Val Tyr Thr Ile Thr Ala Ala
1 5 10 15
Asp Asp Tyr Gln Phe Ser Ser Gln Tyr Gln Pro Gly Gly
20 25
<210>4
<211>26
<212>PRT
＜213〉Avibirnavirus (Avibirnavirus gen.)
<400>4
Trp Ser Ala Arg Gly Ser Leu Ala Val Thr Ile His Gly Gly Asn
1 5 10 15
Tyr Pro Gly Ala Leu Arg Pro Val Thr Leu Val
20 25
Claims (6)
1. chicken infectivity bursa of Fabricius virus IBDV VP2 albumen neutrality B cell antigen epi-position, its aminoacid sequence is Seq No 1:
197H-Cys-Asp-Ser-Ser-Asp-Arg-Pro-Arg-Val-Tyr-Thr-Ile-Thr-OH209。
2. chicken infectivity bursa of Fabricius virus IBDV VP2 albumen neutrality B cell antigen epi-position according to claim 1, according to chicken infectivity bursa of Fabricius virus IBDV VP2 Argine Monohydrochloride sequence extension, the aminoacid sequence after the expansion is Seq No 2 to its aminoacid sequence to the C end:
197H-Cys-Asp-Ser-Ser-Asp-Arg-Pro-Arg-Val-Tyr-Thr-Ile-Thr-Ala-Ala-Asp-Asp-Tyr-Gln-Phe-Ser-Ser-Gln-Tyr-Gln-Pro-Gly-Gly-OH224。
3. according to each described chicken infectivity bursa of Fabricius virus IBDV VP2 albumen neutrality B cell antigen epi-position in claim 1 or 2, it is characterized in that: chicken infectivity bursa of Fabricius virus IBDV VP2 albumen neutrality B cell antigen epi-position is synthetic polypeptide.
4. according to each described chicken infectivity bursa of Fabricius virus IBDV VP2 albumen neutrality B cell antigen epi-position in claim 1 or 2, it is characterized in that: chicken infectivity bursa of Fabricius virus IBDV VP2 albumen neutrality B cell antigen epi-position is carried out chemically modified at N end or C end.
5. chicken infectivity bursa of Fabricius virus IBDV VP2 albumen neutrality B cell antigen epi-position according to claim 4 is characterized in that: described chemically modified is the natural amination or the acetylize of polypeptide chain N end, or the carboxylated naturally or amidation of C end.
6. according to the described chicken infectivity bursa of Fabricius virus IBDVVP2 of each claim albumen neutrality B cell antigen epi-position in the claim 1～2, it is characterized in that: the immunogen of chicken infectivity bursa of Fabricius virus IBDV VP2 albumen neutrality B cell antigen epi-position preparation comprises the described chicken infectivity bursa of Fabricius virus IBDV of each claim VP2 albumen neutrality B cell antigen epitope sequences in the claim 1～4.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5605827A (en) * | 1993-11-04 | 1997-02-25 | The Ohio State University Research Foundation | Infectious bursal disease virus VP2 fusion protein expressed by baculovirus |
| WO1999016866A1 (en) * | 1997-09-30 | 1999-04-08 | University Of Maryland Biotechnology Institute | A method for generating nonpathogenic, infectious birnavirus from synthetic rna transcripts |
| WO2004085634A2 (en) * | 2003-03-24 | 2004-10-07 | Akzo Nobel N.V. | Infectious bursal disease virus mutants and vaccines |
-
2005
- 2005-03-29 CN CNB2005100174536A patent/CN1314706C/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5605827A (en) * | 1993-11-04 | 1997-02-25 | The Ohio State University Research Foundation | Infectious bursal disease virus VP2 fusion protein expressed by baculovirus |
| WO1999016866A1 (en) * | 1997-09-30 | 1999-04-08 | University Of Maryland Biotechnology Institute | A method for generating nonpathogenic, infectious birnavirus from synthetic rna transcripts |
| WO2004085634A2 (en) * | 2003-03-24 | 2004-10-07 | Akzo Nobel N.V. | Infectious bursal disease virus mutants and vaccines |
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