CN105588942A - Genus potyvirus neutralizing epitope identification method and application thereof - Google Patents

Genus potyvirus neutralizing epitope identification method and application thereof Download PDF

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CN105588942A
CN105588942A CN201610070080.7A CN201610070080A CN105588942A CN 105588942 A CN105588942 A CN 105588942A CN 201610070080 A CN201610070080 A CN 201610070080A CN 105588942 A CN105588942 A CN 105588942A
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albumen
epitope
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王永志
苏颖
李启云
李小宇
张春雨
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Jilin Academy of Agricultural Sciences
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Abstract

The invention discloses a genus potyvirus neutralizing epitope identification method and application thereof. The identification method comprises the following steps: S1, preparing materials; S2, cloning genus potyvirus capsid coat protein (CP) genes; S3, constructing a prokaryotic expression vector; S4, identifying recombination CP; S5, purifying the recombination CP; S6, preparing CP monoclonal antibodies and performing virus neutralization test; and S7, performing antigenic epitope analysis.

Description

A kind of potyvirus neutralizing epitope authentication method and application thereof
Technical field
The present invention relates to plant pathology studying technological domain, relate in particular to a kind of marmor upsilonBelong to viral neutralizing epitope authentication method and application thereof.
Background technology
The viroses of plant cause the economic loss of annual 20000000000 dollars. Along with the variation of weather,The change of cropping pattern and the globalization of economic activity, the harm of the viroses of plant increases year by year.Plantibody (Plantibody) is that the restructuring of expressing in plant by genetic engineering means is anti-Body, the application of plantibody technology in plant disease-resistant research is also in the starting stage. About plantingThing virus neutralizing epitope authentication method have not been reported.
Potyvirus (genusPotyvirus) be during fixed plant virus belongs toA large class, 200 kinds of having an appointment are accepted as and determine member or possibility member by ICTV, account for20% of the plant virus sum of having found, and newcomer's quantity is still in continuous increase, is to plantClass at most, the widest, the maximum Tobamovirus of economic implications of host range, represent that strain has large Tofu puddingMosaic virus (SoybeanMosaicVirus, SMV) and marmor upsilon (PotatoVirusY, PVY) etc., the capsid protein (CoatProtein, CP) that its genome end is encoded viral.Soybean Mosaic is the Major Diseases of soybean, not only affects soybean yields and goes to affect soybeanKind, does not also have effectively preventing means at present, and we have proposed a kind of potato Y disease for this reasonPoison belongs to viral neutralizing epitope authentication method and application thereof.
Summary of the invention
The technical problem existing based on background technology, the present invention proposes a kind of marmor upsilonBelong to viral neutralizing epitope authentication method and application thereof.
The present invention propose a kind of potyvirus neutralizing epitope authentication method andApplication, this authentication method comprises the following steps:
S1, prepares material: be ready to e.colistraindh5α, the Rosseta that preserve in laboratoryThe Potyviruses such as bacterial strain, soybean mosaic virus and marmor upsilon represent strain,SP2/0 cell, BALB/c mouse, purchased from the restriction enzyme of the precious biotech firm in Dalian, anti-Transcriptase, T4DNA ligase, EX-TagDNA polymerase, different purchased from GE company of the U.S.Propyl group-β-D-thiogalactoside, Ni2+Ion affinity chromatography post, purchased from Sigma companyThe sheep anti mouse enzyme labelled antibody of HRP mark;
S2, the clone of Potyvirus capsid protein (CP) gene: according to GenBankIn the Auele Specific Primer of existing Potyvirus gene order design CP gene, withThe total RNA of TRIzol method extracting virus infections plant tissue, by 42 DEG C of M-MuLV reverse transcriptaseSynthetic cDNA the first chain of effect 1h, as EX-TaqDNA polymeric enzymatic amplification CP for templateGene, reaction condition is wherein 94-95 DEG C of denaturation 1-3min; 93-95 DEG C of sex change15-20s, 56 DEG C of annealing 30s, 72 DEG C are extended 50s, carry out 30 circulations; 72 DEG C are extended again10min, PCR product reclaims through gel-purified, is connected with pMD18-T carrier, transforms large intestineBacillus DH5 α competent cell, construction recombination plasmid pMD-CP;
S3, the structure of prokaryotic expression carrier: Nco I and Xho I double digestion recombinant plasmid pMD-CPWith prokaryotic expression carrier pET-28a (+), gel-purified reclaims object fragment, by CP gene withPET-28a (+) connects, and construction recombination plasmid pET28a-CP, transforms respectively Escherichia coliDH5 α, cuts after qualification through PCR qualification and enzyme, checks order;
S4, the qualification of recombinant C P albumen: recombinant plasmid pET28a-CP is converted into large intestine barBacterium BL21, the single bacterium colony of picking is identified rear amplification breeding, adopt different IPTG concentration,Induction time, inducing temperature, induction before measurement D600nm value, culture medium antibiotic concentration carry outAbduction delivering, optimizes inductive condition, the table of more different expression vectors and different expression conditionsThe amount of reaching, determines best inductive condition, sets up the expression containing pET-28a (+) empty carrier simultaneouslyBacterium in contrast;
S5, the purifying of recombinant C P albumen: adopt the expression condition of optimizing to carry out great expression,Results thalline, freeze thawing 3 times, with the PBS bacterial sediment that suspends, under the condition of 3-5 DEG CThe centrifugal 9-11min of 4500r/min, inclusion body precipitation washes away major part with 3mol/L urea liquidForeign protein, the centrifugal 10min of 4500r/min under the condition of 4 DEG C, inclusion body precipitation pH=8.0Dissolve 1h containing the lysate room temperature of 8mol/L urea, under the condition of 4 DEG C 12000r/min fromHeart 20min, gets albumin and is combined 0.5-1.5h with Ni2+-NTA filler, use respectively pH=8.0,6.3, after 5.9 8mol/L urea liquid washing foreigh protein removing, with the 8mol/L of pH=4.5Urea liquid wash-out destination protein, the destination protein of collection wash-out, carries out SDS-PAGE qualification;
S6, the preparation of CP protein monoclonal antibody and virus neutralization tests: CP albumen is carried out to listClonal antibody preparation, the monoclonal antibody solution of preparation 1mg/ml concentration; Press 1:20 (gram:Milliliter) get morbidity leaf and mix with PBS solution, make viral suspension, 3000rpm with homogenizerCentrifugal 4-6min, supernatant is virus liquid, and 100ml virus liquid adds 1mg antibody, then logicalCross blade rubbing and connect poison, observe incidence, determine antibody neutralization;
S7, neutralizing epitope analysis: CP albumen is divided into 3 regions, be respectively A, B,C district, approximately 89, each region aa, is divided into two polypeptide by CP albumen, is respectively CP-ABAnd CP-BC, with Bacillus coli expression, with the monoclonal antibody of 10 strain CP albumen respectively withCP-AB and CP-BC test; If react with CP-AB, and do not react with CP-BC, this is describedThe epitope of antibody recognition is positioned at the A district of CP albumen; If all anti-with CP-AB and CP-BCShould, illustrate that the epitope of this antibody recognition is positioned at the B district of CP albumen; If with CP-AB notReaction, and reacts with CP-BC, and epitope that this antibody recognition is described is positioned at CP albumenC district. The epitope of like this, respectively 10 strain monoclonal antibodies being identified is positioned at 3In different regions, navigate to after concrete subregion at definite epitope, should divide and be distinguished intoOverlapped polypeptide, each polypeptide has 16 amino acid residues, has 8 between adjacent polypeptideAmino acid residue overlapping, by elisa assay, is positioned at 16 amino by epitopeWithin acid residue;
Preferably, in described S2, according to existing soybean mosaic virus gene in GenBankThe Auele Specific Primer of sequences Design CP gene, upstream primer5'-atCCATGGatgagattcagaacattga-3'(Nco I), downstream primer5'-atCTCGAGctgtaattggactgcattc-3'(Xho I), with the extracting of TRIzol methodThe total RNA of soybean leaves that soybean mosaic virus infects, with 42 DEG C of effects of M-MuLV reverse transcriptase1h synthesizes cDNA the first chain, as EXTaqDNA polymeric enzymatic amplification CP gene for template,Reaction condition is wherein 94 DEG C of denaturation 2min; 94 DEG C of sex change 15s, 56 DEG C of annealing 30s,72 DEG C are extended 50s, carry out 30 circulations; 72 DEG C are extended 10min again, and PCR product is through gelPurifying reclaims, and is connected with pMD18-T carrier, transforms bacillus coli DH 5 alpha competent cell,Construction recombination plasmid pMD-CP.
Preferably, in described S5, adopt the expression condition of optimizing to carry out great expression, resultsThalline, freeze thawing 3 times, with the PBS bacterial sediment that suspends, 4500r/min under the condition of 4 DEG CCentrifugal 10min, inclusion body precipitation washes away most of foreign protein with 3mol/L urea liquid, at 4 DEG CCondition under the centrifugal 10min of 4500r/min, inclusion body precipitation with pH=8.0 containing 8mol/L urineThe lysate room temperature of element is dissolved 1h, the centrifugal 20min of 12000r/min under the condition of 4 DEG C,Get albumin and be combined 1h with Ni2+-NTA filler, use respectively pH=8.0,6.3,5.9After 8mol/L urea liquid washing foreigh protein removing, wash with the 8mol/L urea liquid of pH=4.5De-destination protein, the destination protein of collection wash-out, carries out SDS-PAGE qualification.
Preferably, in described S6, CP albumen is carried out to monoclonal antibody preparation, by 1:20Get morbidity leaf and mix with PBS solution, make viral suspension with homogenizer, 3000rpm is centrifugal5min, supernatant is virus liquid, and 100ml virus liquid adds 1mg antibody, then passes through bladeFriction connects poison, observes incidence.
Brief description of the drawings
Fig. 1 is a kind of potyvirus neutralizing epitope authentication method that the present invention proposesAnd the CP protein fraction of application is expressed and the synthetic schematic diagram of polypeptide;
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is done to further explanation.
The present invention propose a kind of potyvirus neutralizing epitope authentication method andApplication, this authentication method comprises the following steps:
S1, prepares material: be ready to e.colistraindh5α, the Rosseta that preserve in laboratoryBacterial strain, No. 3, No. 1 strain of soybean mosaic virus and SP2/0 cell, pick up from the Inner Mongol and prick blueThe potato sample that the marmor upsilon in village infects, is buied by Changchun institute of Biological ProductsBALB/c mouse, purchased from restriction enzyme, reverse transcriptase, the T4DNA of the precious biotech firm in DalianLigase, EX-TagDNA polymerase, purchased from isopropyl-β-D-sulfo-of GE company of the U.S.Galactoside, Ni2+Ion affinity chromatography post, purchased from the goat-anti of the HRP mark of Sigma companyMouse enzyme labelled antibody;
S2, the clone of soybean mosaic virus CP gene: according to existing soybean in GenBankThe Auele Specific Primer of mosaic virus gene order design CP gene, upstream primer5'-atCCATGGatgagattcagaacattga-3'(Nco I), downstream primer5'-atCTCGAGctgtaattggactgcattc-3'(Xho I), with the extracting of TRIzol methodThe total RNA of soybean leaves that soybean mosaic virus infects, with 42 DEG C of effects of M-MuLV reverse transcriptase1h synthesizes cDNA the first chain, as TaqplusDNA polymeric enzymatic amplification CP base for templateCause, reaction condition is wherein 93-95 DEG C of denaturation 1-3min; 93-95 DEG C of sex change 14-16s,56 DEG C of annealing 30s, 72 DEG C are extended 50s, carry out 30 circulations; 72 DEG C are extended 10min again,PCR product reclaims through gel-purified, is connected with pMD18-T carrier, transforms bacillus coli DH 5 alphaCompetent cell, construction recombination plasmid pMD-CP;
S3, the structure of prokaryotic expression carrier: Nco I and Xho I double digestion recombinant plasmid pMD-CPWith prokaryotic expression carrier pET-28a (+), gel-purified reclaims object fragment, by the CP of SMVGene is connected with pET-28a (+), and construction recombination plasmid pET28a-CP transforms respectively large intestine barBacterium DH5 α, cuts after qualification through PCR qualification and enzyme, checks order;
S4, the qualification of restructuring SMVCP albumen: recombinant plasmid pET28a-CP is converted into greatlyEnterobacteria BL21, the single bacterium colony of picking is identified rear amplification breeding, adopts different IPTGConcentration, induction time, inducing temperature, bacterial density, culture medium antibiotic concentration are inducedExpress, optimize inductive condition, the expression of more different expression vectors and different expression conditions,Determine best inductive condition, set up the expression bacterium conduct containing pET-28a (+) empty carrier simultaneouslyContrast;
S5, the purifying of restructuring SMVCP albumen: adopt the expression condition of optimizing to carry out large scaleReach, results thalline, freeze thawing 3 times, with the PBS bacterial sediment that suspends, under the condition of 3-5 DEG CThe centrifugal 9-11min of 4500r/min, inclusion body precipitation washes away major part with 3mol/L urea liquidForeign protein, the centrifugal 10min of 4500r/min under the condition of 4 DEG C, inclusion body precipitation pH=8.0Dissolve 1h containing the lysate room temperature of 8mol/L urea, under the condition of 4 DEG C 12000r/min fromHeart 20min, gets albumin and is combined 0.5-1.5h with Ni2+-NTA filler, use respectively pH=8.0,6.3, after 5.9 8mol/L urea liquid washing foreigh protein removing, with the 8mol/L of pH=4.5Urea liquid wash-out destination protein, the destination protein of collection wash-out, carries out SDS-PAGE qualification;
S6, virus neutralization tests: CP albumen is carried out to monoclonal antibody preparation, by 1:20Get morbidity leaf and mix with PBS solution, make viral suspension with homogenizer, 3000rpm is centrifugal4-6min, supernatant is virus liquid, and 100ml virus liquid adds 1mg antibody, then passes through leafSheet friction connects poison, observes incidence;
S7, interpretation of result: with reference to Fig. 1, CP albumen is divided into 3 regions, be respectively A,B, C district, approximately 89, each region aa, is divided into two polypeptide by CP albumen, is respectively CP-ABAnd CP-BC, with Bacillus coli expression, with the monoclonal antibody of 10 strain CP albumen respectively withCP-AB and CP-BC test; If react with CP-AB, and do not react with CP-BC, this is describedThe epitope of antibody recognition is positioned at the A district of CP albumen; If all anti-with CP-AB and CP-BCShould, illustrate that the epitope of this antibody recognition is positioned at the B district of CP albumen; If with CP-AB notReaction, and reacts with CP-BC, and epitope that this antibody recognition is described is positioned at CP albumenC district. The epitope of like this, respectively 10 strain monoclonal antibodies being identified is positioned at 3In different regions;
As shown above, navigate to after concrete subregion at definite epitope, should divide and be distinguished into phasePolypeptide is folded in mutual respect, and each polypeptide has 16 amino acid residues, has 8 ammonia between adjacent polypeptideBase acid residue overlapping, by elisa assay, is positioned at 16 amino acid by epitopeWithin residue;
Clone's soybean mosaic virus CP gene is inserted to prokaryotic expression carrier pET-28a (+)In, obtain recombinant expression plasmid pET28a-CP, transform Escherichia coli with pET28a-CPBL21 (DE3), under the induction of IPTG, gives expression to CP albumen, through affinity chromatography, obtainsThe CP albumen of purifying.
With reference to following table, taking the CP albumen of purifying as antigen coated ELISA Plate, on fused cellCarry out clearly indirect ELISA screening and detect, record altogether 57 strong positive holes. Infect with SMVThe positive antigen of soybean leaves total protein is taking healthy soybean leaves total protein as contrast antigen, logicalCross indirect ELISA, the cells and supernatant in these 57 positive holes is further screened, determineThe antibody of 10 strain of hybridoma secretions is natural viral CP protein-specific body, to this 10Strain cell carries out 5 monoclonals of taking turns limiting dilution, finally obtains 10 strains and can secrete CP eggThe hybridoma cell line of white monoclonal antibody, respectively called after 1C2,2C1,2A8,2D3,3B8,4F9,4G12,5D2,6E7,6E10, use Sigma company mouse-anti body SubtypesKit, to the secreted 10 strain monoclonal antibody 1C2 of 10 strain of hybridoma that obtain,
2C1,2A8,2D3,3B8,4F9,4G12,5D2,6E7,6E10 carry out hypotype and divideAnalyse, result shows, monoclonal antibody 1C2,2C1,2D3,4F9,4G12,5D2,6E7,6E10 is IgG1 hypotype, and 2A8 and 3B8 are IgG2b hypotypes, potted plant neutralization test and largeTanaka and result of the test show, 10 strain monoclonal antibodies all in tool and active, all belong to neutralizationAntibody, can block SMV and infect, and the neutralization of different neutralizing antibodies is active different, wherein 6E7,The neutralization activity of 1C2,2A8 is the strongest, and the neutralization activity of 2D3,3B8,2C1,5D2 is medium,The neutralization activity of 4G12,6E10,4F9 slightly a little less than, find monoclonal antibody by epitope analysisThe common Recognition polypeptide CP-A1 of 2A8,3B8 and 5D2 and CP-A2, and peptide C P-A1 and CP-A2It is identical having the sequence of 1 section of 8 amino acid residue, i.e. sequence MDAGNDPK, therefore,An epi-position MDAGNDPK of the common identification of monoclonal antibody 2A8,3B8 and 5D2, called afterCP-A1A2; Monoclonal antibody 6E7 Recognition polypeptide CP-A2, sequence is MDAGNDPKKSTSSSKG,Called after epi-position CP-A2; Monoclonal antibody 4G12 Recognition polypeptide CP-B5, sequence isANGVWVMMDGEEQIEY, called after epi-position CP-B5; Monoclonal antibody 6E10 Recognition polypeptideCP-B6, sequence is DGEEQIEYPLKPIVEN, called after epi-position CP-B6. In addition, Dan KeThe A district of grand antibody 2C1 and 1C2 identification CP albumen, knows but polypeptide scanning can not find itOther polypeptide, illustrates that the epi-position of 2C1 and 1C2 identification is not linear epitope, but comformational epitope,We distinguish called after CP-AX1 and CP-AX2, between comformational epitope CP-AX1 and CP-AX2May be identical, also may be different; Equally, monoclonal antibody 4F9 and 2D3 identification CP albumenThe comformational epitope in B district, our called after CP-BX1 and CP-BX2, comformational epitope CP-BX1And may be identical between CP-BX2, also may be different.
Sequence analysis shows, neutralizing epitope CP-A1A2 (AA9-16) and CP-A2 (AA9-24)In No. 1, No. 3 strains of soybean mosaic virus, all guard northeastward, but in national orderRow are middle discovery relatively, and these two epi-positions are not guarded between different strains very much, and neutralizeEpi-position CP-B5 (AA124-139) and CP-B6 (AA132-147) are known used domesticIn strain sequence, all guard, find with the SMVCP Gene sequence comparison of abroad having reported,The sequence complete one of neutralizing epitope CP-B5 (AA124-139) and CP-B6 (AA132-147)Cause.
Gathered the Ma Ling that marmor upsilon infects from potato planting district, Zhalantun, the Inner MongolPotato leaf sample, taking incidence of leaf total protein as antigen, taking healthy leaves as contrast, ELISAAnalysis result shows, monoclonal antibody 6E10 and 4G12 can identify potato Y disease, and2A8,3B8,5D2 and 6E7 can not identify marmor upsilon.
In order to illustrate SMVCP albumen high conservative epitope CP-B5 and CP-B6 at Ma LingConservative during potato Y virus belongs to, we have chosen 30 in Potyvirus at randomVirus sequence, with SMVCP protein sequence and CP-B5 and the CP-B6 epi-position of our mensurationSequence compares, and result shows the homology between Potyvirus CP albumen complete sequenceBetween 50.2%-61.7%, average out to 55.9%; CP-B5 and CP-B6 epitope sequences sameSource property is 62.5%-95.8%, average 77.5%, higher by 21.6 than the homology of CP albumen complete sequencePercentage point, therefore epitope CP-B5 and CP-B6 are the conservatives of PotyvirusEpitope.
Potyvirus is a Tobamovirus maximum in plant virus, and member is numerous, itsEconomic implications is also maximum, and its genome is RNA, very easily variation. This research has been found 2 greatlyBean mosaic virus neutralizing epitope is high conservative in Potyvirus, and this also indicatesThe importance of this sequence in function, this epi-position can be used as the target spot of disease-resistant research. Therefore,The qualification of this epi-position, has important theory and realistic meaning.
The resistance being mediated by viral gene, owing to recombinating with natural strain, exists biologicalSecurity risk, is difficult to actual applying. Concerning RNA virus, due to viral height variationProperty, utilize RNA to interfere the resistance of mediation usually to there is strain specificity, and do not there is broad spectrum activity.The target spot of plantibody is protein, the conservative of albumen is more much higher than RNA, especially albumenKey function region, often high conservative, the CP albumen neutralizing epitope CP-B5 of this research qualificationWith CP-B6 be exactly 2 very conservative functional areas.
Antibody is the composition often having in food, especially, in dairy produce and egg food, all containsA large amount of antibody, has been eaten several thousand by the mankind, fact proved antibody component in foodHarmless, and in the baby food of some colostrum classes, antibody is as beneficiating ingredientExist. Plantibody is the antibody by expression of plants, can reduce the production cost of antibody,The output that increases antibody, finally some plantibodies are applied to mankind's medical treatment, therefore, Genes For Plant ToleranceBody is also harmless to human body.
The above, be only preferably detailed description of the invention of the present invention, but protection model of the present inventionEnclose and be not limited to this, any be familiar with those skilled in the art the present invention disclose skillWithin the scope of art, be equal to replacement or changed according to technical scheme of the present invention and inventive concept thereofBecome, within all should being encompassed in protection scope of the present invention.

Claims (2)

1. potyvirus neutralizing epitope authentication method and an application thereof, its spyLevy and be, this authentication method comprises the following steps:
S1, prepares material: be ready to e.colistraindh5α, the Rosseta that preserve in laboratoryThe Potyviruses such as bacterial strain, soybean mosaic virus and marmor upsilon represent strain,SP2/0 cell, BALB/c mouse, purchased from the restriction enzyme of the precious biotech firm in Dalian, anti-Transcriptase, T4DNA ligase, EX-TagDNA polymerase, different purchased from GE company of the U.S.Propyl group-β-D-thiogalactoside, Ni2+Ion affinity chromatography post, purchased from Sigma companyThe sheep anti mouse enzyme labelled antibody of HRP mark;
S2, the clone of Potyvirus capsid protein (CP) gene: according to GenBankIn the Auele Specific Primer of existing Potyvirus gene order design CP gene, withThe total RNA of TRIzol method extracting virus infections plant tissue, by 42 DEG C of M-MuLV reverse transcriptaseSynthetic cDNA the first chain of effect 1h, as EX-TaqDNA polymeric enzymatic amplification CP for templateGene, reaction condition is wherein 94-95 DEG C of denaturation 1-3min; 93-95 DEG C of sex change15-20s, 56 DEG C of annealing 30s, 72 DEG C are extended 50s, carry out 30 circulations; 72 DEG C are extended again10min, PCR product reclaims through gel-purified, is connected with pMD18-T carrier, transforms large intestineBacillus DH5 α competent cell, construction recombination plasmid pMD-CP;
S3, the structure of prokaryotic expression carrier: Nco I and Xho I double digestion recombinant plasmid pMD-CPWith prokaryotic expression carrier pET-28a (+), gel-purified reclaims object fragment, by CP gene withPET-28a (+) connects, and construction recombination plasmid pET28a-CP, transforms respectively Escherichia coliDH5 α, cuts after qualification through PCR qualification and enzyme, checks order;
S4, the qualification of recombinant C P albumen: recombinant plasmid pET28a-CP is converted into large intestine barBacterium BL21, the single bacterium colony of picking is identified rear amplification breeding, adopt different IPTG concentration,
Induction time, inducing temperature, bacterial concentration, culture medium antibiotic concentration carry out induction tableReach, optimize inductive condition, the expression of more different expression vectors and different expression conditions,Determine best inductive condition, set up the expression bacterium conduct containing pET-28a (+) empty carrier simultaneouslyContrast;
S5, the purifying of recombinant C P albumen: adopt the expression condition of optimizing to carry out great expression,Results thalline, freeze thawing 3 times, with the PBS bacterial sediment that suspends, under the condition of 3-5 DEG CThe centrifugal 9-11min of 4500r/min, inclusion body precipitation washes away major part with 3mol/L urea liquidForeign protein, the centrifugal 10min of 4500r/min under the condition of 4 DEG C, inclusion body precipitation pH=8.0Dissolve 1h containing the lysate room temperature of 8mol/L urea, under the condition of 4 DEG C 12000r/min fromHeart 20min, gets albumin and is combined 0.5-1.5h with Ni2+-NTA filler, use respectively pH=8.0,6.3, after 5.9 8mol/L urea liquid washing foreigh protein removing, with the 8mol/L of pH=4.5Urea liquid wash-out destination protein, the destination protein of collection wash-out, carries out SDS-PAGE qualification;
S6, the preparation of CP protein monoclonal antibody and virus neutralization tests: CP albumen is carried out to listClonal antibody preparation, the monoclonal antibody solution of preparation 1mg/ml concentration; Press 1:20 (gram:Milliliter) get morbidity leaf and mix with PBS solution, make viral suspension, 3000rpm with homogenizerCentrifugal 4-6min, supernatant is virus liquid, and 100ml virus liquid adds 1mg antibody, then logicalCross blade rubbing and connect poison, observe incidence, determine antibody neutralization;
S7, neutralizing epitope analysis: CP albumen is divided into 3 regions, be respectively A, B,C district, approximately 89, each region aa, is divided into two polypeptide by CP albumen, is respectively CP-ABAnd CP-BC, with Bacillus coli expression, with the monoclonal antibody of CP albumen respectively with CP-ABTest with CP-BC; If react with CP-AB, and do not react with CP-BC, this antibody knowledge is describedOther epitope is positioned at the A district of CP albumen; If all react with CP-AB and CP-BC, sayThe epitope of bright this antibody recognition is positioned at the B district of CP albumen; If do not react with CP-AB,And react with CP-BC, illustrate that the epitope of this antibody recognition is positioned at the C district of CP albumen.The epitope of like this, respectively 10 strain monoclonal antibodies being identified be positioned at 3 differentIn region, navigate to after concrete subregion at definite epitope, should divide and be distinguished into mutual weightFolded polypeptide, each polypeptide has 16 amino acid residues, has 8 amino acid between adjacent polypeptideResidue overlapping, by elisa assay, is positioned at 16 amino acid residues by epitopeWithin.
2. a kind of potyvirus neutralizing epitope mirror according to claim 1Determine method and application thereof, it is characterized in that, in described S7, CP albumen is divided into 3 districtsTerritory, is respectively A, B, C district, and approximately 89, each region amino acid, is divided into two by CP albumenIndividual polypeptide, is respectively CP-AB and CP-BC, with Bacillus coli expression, active with having neutralizationCP albumen monoclonal antibody respectively with CP-AB and CP-BC test; If anti-with CP-ABShould, and not reacting with CP-BC, epitope that this antibody recognition is described is positioned at CP albumenA district; If all react with CP-AB and CP-BC, illustrate that the epitope of this antibody recognition is positioned atThe B district of CP albumen; If do not react with CP-AB, and react with CP-BC, this antibody is describedThe epitope of identification is positioned at the C district of CP albumen. Like this, respectively by 10 strain monoclonal antibodiesThe epitope of identifying is positioned in 3 different regions, navigates at definite epitopeAfter concrete subregion, should divide and be distinguished into overlapped polypeptide, each polypeptide has 16 aminoAcid residue, has the overlapping of 8 amino acid residues between adjacent polypeptide, by elisa assay,Within epitope is positioned to 16 amino acid residues.
CN201610070080.7A 2016-02-01 2016-02-01 Genus potyvirus neutralizing epitope identification method and application thereof Pending CN105588942A (en)

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