CN102206253B - Actin depolymerizing factor epitope, actin depolymerizing factor antibody and use thereof - Google Patents
Actin depolymerizing factor epitope, actin depolymerizing factor antibody and use thereof Download PDFInfo
- Publication number
- CN102206253B CN102206253B CN 201010520323 CN201010520323A CN102206253B CN 102206253 B CN102206253 B CN 102206253B CN 201010520323 CN201010520323 CN 201010520323 CN 201010520323 A CN201010520323 A CN 201010520323A CN 102206253 B CN102206253 B CN 102206253B
- Authority
- CN
- China
- Prior art keywords
- depolymerizing factor
- actin depolymerizing
- antibody
- actin
- epitope
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 108090000082 Destrin Proteins 0.000 title claims abstract description 70
- 108090001064 Gelsolin Proteins 0.000 title claims abstract description 70
- 102000003668 Destrin Human genes 0.000 title claims abstract description 67
- 238000002360 preparation method Methods 0.000 claims abstract description 24
- 239000000203 mixture Substances 0.000 claims abstract description 19
- 238000000034 method Methods 0.000 claims abstract description 14
- 235000007164 Oryza sativa Nutrition 0.000 claims description 29
- 235000009566 rice Nutrition 0.000 claims description 28
- 238000001514 detection method Methods 0.000 claims description 11
- 239000002671 adjuvant Substances 0.000 claims description 8
- 210000004369 blood Anatomy 0.000 claims description 8
- 239000008280 blood Substances 0.000 claims description 8
- 108010045069 keyhole-limpet hemocyanin Proteins 0.000 claims description 7
- 241001465754 Metazoa Species 0.000 claims description 6
- 239000003795 chemical substances by application Substances 0.000 claims description 4
- 230000036039 immunity Effects 0.000 claims description 3
- 240000007594 Oryza sativa Species 0.000 claims description 2
- 239000005018 casein Substances 0.000 claims description 2
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 claims description 2
- 235000021240 caseins Nutrition 0.000 claims description 2
- 238000000746 purification Methods 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 1
- 241000209094 Oryza Species 0.000 description 27
- 108090000623 proteins and genes Proteins 0.000 description 21
- 102000004169 proteins and genes Human genes 0.000 description 19
- 235000018102 proteins Nutrition 0.000 description 16
- 108010085238 Actins Proteins 0.000 description 12
- 102000007469 Actins Human genes 0.000 description 12
- 239000000427 antigen Substances 0.000 description 10
- 102000036639 antigens Human genes 0.000 description 10
- 108091007433 antigens Proteins 0.000 description 10
- 108090000765 processed proteins & peptides Proteins 0.000 description 10
- 230000003053 immunization Effects 0.000 description 8
- 238000002649 immunization Methods 0.000 description 8
- 210000003205 muscle Anatomy 0.000 description 7
- 241000283973 Oryctolagus cuniculus Species 0.000 description 6
- 241000196324 Embryophyta Species 0.000 description 5
- 210000002966 serum Anatomy 0.000 description 5
- 210000001519 tissue Anatomy 0.000 description 5
- 210000003462 vein Anatomy 0.000 description 5
- 102000017734 ADF/Cofilin Human genes 0.000 description 4
- 108050005951 ADF/Cofilin Proteins 0.000 description 4
- 150000001413 amino acids Chemical class 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 229920001184 polypeptide Polymers 0.000 description 4
- 102000004196 processed proteins & peptides Human genes 0.000 description 4
- 238000001262 western blot Methods 0.000 description 4
- 230000000890 antigenic effect Effects 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 239000000284 extract Substances 0.000 description 3
- 239000000835 fiber Substances 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 238000001819 mass spectrum Methods 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- 102000002151 Microfilament Proteins Human genes 0.000 description 2
- 108010040897 Microfilament Proteins Proteins 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 239000006180 TBST buffer Substances 0.000 description 2
- WNROFYMDJYEPJX-UHFFFAOYSA-K aluminium hydroxide Chemical compound [OH-].[OH-].[OH-].[Al+3] WNROFYMDJYEPJX-UHFFFAOYSA-K 0.000 description 2
- 229910021502 aluminium hydroxide Inorganic materials 0.000 description 2
- 235000001014 amino acid Nutrition 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 239000000539 dimer Substances 0.000 description 2
- 238000003119 immunoblot Methods 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000009413 insulation Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 210000003632 microfilament Anatomy 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000011587 new zealand white rabbit Methods 0.000 description 2
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 102000015693 Actin Depolymerizing Factors Human genes 0.000 description 1
- 108010038798 Actin Depolymerizing Factors Proteins 0.000 description 1
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 1
- 238000009010 Bradford assay Methods 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 244000050510 Cunninghamia lanceolata Species 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 241000237988 Patellidae Species 0.000 description 1
- 241000276498 Pollachius virens Species 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 108010008038 Synthetic Vaccines Proteins 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 210000001367 artery Anatomy 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 238000010009 beating Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000007622 bioinformatic analysis Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 210000001715 carotid artery Anatomy 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000012916 chromogenic reagent Substances 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 230000009514 concussion Effects 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- ATDGTVJJHBUTRL-UHFFFAOYSA-N cyanogen bromide Chemical compound BrC#N ATDGTVJJHBUTRL-UHFFFAOYSA-N 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 210000004292 cytoskeleton Anatomy 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 108060003552 hemocyanin Proteins 0.000 description 1
- 239000000568 immunological adjuvant Substances 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 210000002220 organoid Anatomy 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000007026 protein scission Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 235000011149 sulphuric acid Nutrition 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Images
Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention belongs to the field of molecular biology and immunology and relates to an actin depolymerizing factor epitope, an actin depolymerizing factor antibody and use thereof. Particularly, the actin depolymerizing factor epitope has a sequence shown by SEQ ID No.3. The invention also relates to an actin depolymerizing factor polyclonal antibody, which is specifically combined with the epitope. The polyclonal antibody has high specificity and titer. The invention also relates to the preparation method and use of the polyclonal antibody, a composition containing the polyclonal antibody,and a method for detecting the actin depolymerizing factor.
Description
Technical field
The invention belongs to molecular biology and field of immunology.Particularly, relate to a kind of actin depolymerizing factor epitope, anti-actin depolymerizing factor antibody, the invention still further relates to the composition and the purposes of described antibody in detecting actin depolymerizing factor that contain described epitope and antibody respectively.
Background technology
(actin depolymerizing factor/cofilin ADF/cofilin) belongs to actin binding protein (actin-binding protein) family to actin depolymerizing factor.Up to now, can in all eukaryotic cells, detect ADF/cofilin.They can regulate (fibre) actin filament cytoskeleton (F-actin cytoskeleton), influence the various physiological functions of cell.Different biologies has different ADF/cofilin, but its function is similar substantially.ADF/cofilin can make (fibre) actin filament (F-actin) depolymerize, and this activity that depolymerizes is reversible, ADF/cofllin cuts F-actin and can improve the ability that globular actin (G-actin) leaves fiber overhang (pointed end), and its effect is regulated and control by several factors.Actin muscle to biological normal cell form keep, the apical growth of division of cytoplasm, cyclosis, organoid motion and plant plays an important role.In case destroy the running balance of biological cell Actin muscle, biology more or less shows paramophia.
Studies show that, actin depolymerizing factor depolymerization and cutting Actin muscle, the running balance that can destroy Actin muscle is expressed in crossing of actin depolymerizing factor.In a single day Actin muscle running balance destroyed, and just might influence plant normal growth.The dynamic change of actin depolymerizing factor in plant materials is the important factor that influences the running balance of Actin muscle.
Paddy rice (Oryza sativa L.) is one of most important food crop in the world, also is the model plant of fundamental researchs such as growth and development of plants, adverse circumstance reaction.The achievement of rice genome examining order has greatly promoted the progress of paddy rice correlative study, screens and proposed suitable rice actin depolymerizing factor gene actin depolymerizing factor.Actin depolymerizing factor gene Os07t0484200-01 by bioinformatic analysis, is positioned on the 7th karyomit(e) of paddy rice, dna sequence dna total length 1514bp, cDNA sequence total length 420bp, the actin depolymerizing factor albumen that 139 amino acid of encoding are formed.By RAP-DB database compare of analysis, actin depolymerizing factor Os07t0484200-01 is a kind of actin depolymerizing factor albumen.At present also not about relevant reports such as this gene and albumen.
At present the research of the antibody that actin depolymerizing factor total length antigen is produced be blank.Consulting document finds, the probability that BEPITOPE software has above 30% can not predict antigenic determinant (Chang HT, Liu CH, Pai TW.Estimation and extraction of B-cell linear epitopes predicted by mathematical morphology approaches.J Mol Recognit.2008 Nov-Dec; 21 (6): 431-41), this software exists the specificity problem to the antigenic determinant prediction of total length antigen.
Summary of the invention
In order to address the above problem, the inventor is obtaining by the BEPITOPE software prediction on the basis of a plurality of epitope sequences, a large amount of experiments and unremitting effort have been carried out, found that it is effective antigens epi-position that an epitope sequence C KFKFQELKTRRG (SEQID NO:3) is arranged in 36 epitopes of prediction, and it has good antigen-specific.Following invention is provided thus:
One aspect of the present invention relates to a kind of actin depolymerizing factor epitope, and its aminoacid sequence is shown in SEQ ID NO:3.
Epitope of the present invention can obtain by the peptide synthetic technology chemosynthesis of routine, also can express obtaining in suitable host; Preferably chemosynthesis.
Of the present inventionly also relate in one aspect to a kind of composition, it comprises the actin depolymerizing factor epitope shown in the SEQ ID NO:3, randomly, can contain immunological adjuvant, for example aluminium hydroxide, Freund's complete adjuvant or Freund's incomplete adjuvant etc.
Invention also relate in one aspect to a kind of actin depolymerizing factor epitope-carrier complexes; Wherein, described actin depolymerizing factor epitope is the actin depolymerizing factor epitope shown in the SEQ ID NO:3, and described carrier can be keyhole limpet hemocyanin (KLH), BSA or casein etc.
Of the present inventionly also relate in one aspect to a kind of anti-actin depolymerizing factor antibody, described anti-actin depolymerizing factor antibody can be specifically in conjunction with the actin depolymerizing factor epitope shown in the SEQ ID NO:3.Described anti-actin depolymerizing factor antibody can be monoclonal antibody, also can be polyclonal antibody.
Anti-actin depolymerizing factor polyclonal antibody of the present invention can derive from the animals of various conventional preparation polyclonal antibodies, goat for example, rabbit, rat, mouse etc.
To those skilled in the art, can prepare monoclonal antibody according to the actin depolymerizing factor epitope shown in the SEQ ID NO:3, concrete operations can be referring to the technical manual of this area, also can reference Nature 1975 Kohler﹠amp for example; Milstein Vol256, p495.
Of the present inventionly also relate in one aspect to a kind of serum (abbreviation polyvalent antibody) that contains anti-actin depolymerizing factor polyclonal antibody, it makes by using the epitope immune animal shown in the SEQ ID NO:3.
Of the present inventionly also relate in one aspect to a kind of anti-actin depolymerizing factor preparation method of polyclonal antibody, comprise the step of the actin depolymerizing factor epitope shown in the SEQ ID NO:3 as antigen-immunized animal.Randomly, described immune step can add adjuvant, for example aluminium hydroxide, Freund's complete adjuvant or Freund's incomplete adjuvant, etc.
In one embodiment of the invention, described anti-actin depolymerizing factor preparation method of polyclonal antibody comprises the steps:
1) with the actin depolymerizing factor epitope shown in the SEQ ID NO:3 as antigen-immunized animal;
2) get blood, centrifugal collection polyvalent antibody; With
3) purification step 2) in polyvalent antibody, obtain anti-actin depolymerizing factor polyclonal antibody.
Of the present inventionly also relate in one aspect to a kind of composition, it comprises anti-actin depolymerizing factor polyclonal antibody of the present invention.
Of the present inventionly also relate in one aspect to a kind of actin depolymerizing factor detection agent, it comprises anti-actin depolymerizing factor polyclonal antibody of the present invention.
(SEQ ID NO:2) is as follows for the aminoacid sequence of actin depolymerizing factor:
MANSASGLAVNDE
FRFIVFKIDDKAMEIKVERLGQTAEGYEDFAATLPADECRYAVYDLDFVTDENCQKSKIFFFSWSPDTARTRSKMLYASSKDRFRRELDGIQCEIQATDPSEMSLDIIRARAH(SEQ?ID?NO：2)
The sequence that wherein adds frame is SEQ ID NO:3
The purposes of anti-actin depolymerizing factor polyclonal antibody of the present invention in the medicine of preparation detection actin depolymerizing factor that also relate in one aspect to of the present invention.
Of the present inventionly also relate in one aspect to a kind of method that detects actin depolymerizing factor, described method comprises the step of using anti-actin depolymerizing factor polyclonal antibody of the present invention.Particularly, comprise the steps:
1) testing sample and anti-actin depolymerizing factor polyclonal antibody of the present invention are hatched, the actin depolymerizing factor specificity of described anti-actin depolymerizing factor polyclonal antibody in testing sample is combined, form immunocomplex thus; With
2) detect whether there is immunocomplex.
The method of above-mentioned detection actin depolymerizing factor can detect the having or not of actin depolymerizing factor, it is carried out sxemiquantitative (for example western blot method); Under the situation that the actin depolymerizing factor standard substance are arranged, can also carry out detection by quantitative to actin depolymerizing factor by the ELISA method.
In one embodiment of the invention, described actin depolymerizing factor is the actin depolymerizing factor of paddy rice.Concrete, described paddy rice is paddy rice 93-11.
The beneficial effect of the invention
1) antigen obtains conveniently.The present invention adopts the method for chemically synthesized polypeptide to obtain antigen, has synthesized 13 amino acid whose antigenic determinants, has guaranteed the specificity of antibody, has avoided the loaded down with trivial details of antigen prepd.
2) Antibody Preparation is simple, and harvest yield is big.Get synthetic antigen 1-2mg immunity new zealand white rabbit, every 14 days booster immunizations once; 7 days ear veins are got blood behind the 2nd booster immunization, and centrifugal collection polyvalent antibody obtains a large amount of polyclonal antibodies.
3) high specificity of antibody.With the polyclonal antibody high specificity that antigen immune obtains, the information of tiring of its polyvalent antibody is KLH＞25600, naked peptide＞12800, antibody and antigen in conjunction with sensitive, for the expression of scientific research actin depolymerizing factor albumen provides solid basis.
Description of drawings
Fig. 1: use anti-actin depolymerizing factor polyclonal antibody to the immunoblotting detected result of actin depolymerizing factor at the paddy rice different tissues.
Fig. 2: the one-level mass spectrum of the actin depolymerizing factor of how anti-detection.
Fig. 3: the second order ms figure of the actin depolymerizing factor of how anti-detection.
Embodiment
Be described in detail below in conjunction with the embodiment of the present invention of embodiment.It will be understood to those of skill in the art that the following examples only are used for explanation the present invention, and should not be considered as limiting scope of the present invention.Unreceipted concrete technology or condition person among the embodiment, according to the described technology of the document in this area or condition (for example with reference to works such as J. Sa nurse Brookers, " the molecular cloning experiment guide " that Huang Peitang etc. translate, the third edition, Science Press) or carry out according to product description.The unreceipted person of production firm of agents useful for same or instrument, being can be by the conventional products of commercial acquisition.
Embodiment 1: the prediction of candidate antigens epi-position 1
The gene of rice actin depolymerizing factor correspondence is Os07g0485100, GenBank accession number: NP_001059648.1.The reading frame sequence is as follows:
ATGGCGAATTCTGCGTCAGGGCTGGCGGTGAACGACGAGTGCAAGTTCAAGTTCCAGGAGCTGAAGACGAGGAGGGGGTTCAGGTTCATCGTGTTCAAGATCGACGACAAGGCCATGGAGATCAAGGTGGAGAGGCTCGGGCAGACTGCCGAGGGCTACGAGGACTTCGCCGCCACCCTCCCCGCCGACGAGTGCCGCTACGCCGTCTACGACCTCGACTTCGTCACCGACGAGAACTGCCAGAAGAGCAAGATCTTCTTCTTCTCCTGGTCGCCTGACACGGCGAGGACAAGGAGCAAGATGCTGTACGCGAGCTCCAAGGACAGGTTCAGGAGGGAGCTGGACGGAATCCAGTGCGAGATTCAGGCCACAGACCCCAGCGAGATGAGCCTCGACATCATCAGAGCCAGAGCTCACTGA(SEQ?ID?NO：1)
Coded rice actin depolymerizing factor full length sequence is as follows:
MANSASGLAVNDECKFKFQELKTRRGFRFIVFKIDDKAMEIKVERLGQTAEGYEDFAATLPADECRYAVYDLDFVTDENCQKSKIFFFSWSPDTARTRSKMLYASSKDRFRRELDGIQCEIQATDPSEMSLDIIRARAH(SEQID?NO：2)
Then according to SEQ ID NO:2, with BEPITOPE software the protein of rice actin depolymerizing factor genes encoding is carried out the prediction of epitope.Five kinds of method Standard, the Karplus, Emini, Amphiphi, the Pellequer that have used BEPITOPE software to provide in the present embodiment, and the integrated approach cons_Sta_Kar_Emi_Amp_Pel of these five kinds of methods, all parameters are selected acquiescence.Concrete grammar can be with reference to Odorico M, Pellequer J L.BEPITOPE:predicting the location of continuous epitopes and patterns in proteins[J] .J Mol Recognit, 2003,16 (1): 20-22.
Prediction has obtained 12 candidates' epitope altogether, carries out selecting target fragment after uniqueness detects with the rice protein of BLASTP storehouse, and its peptide sequence of selecting is: CKFKFQELKTRRG.Then with above-mentioned fragment in the rice protein storehouse (RAP-DB database, network address: http://rapdb.dna.affrc.go.jp/) carry out uniqueness retrieval (protein sequence comparison), determined the uniqueness of this fragment in the rice protein storehouse.
Embodiment 2: the chemosynthesis of epitope 1
Peptide sequence shown in the SEQ ID NO:3 is carried out chemosynthesis (synthetic by the gill biochemical corp), obtain the epitope 1 of actin depolymerizing factor.
Embodiment 3: the preparation of epitope 1-KLH mixture
Adopt the glutaraldehyde connection method, the N end of epitope 1 synthetic among the embodiment 2 is crosslinked with crosslinked carrier proteins-keyhole limpet hemocyanin (KLH), obtain epitope 1-KLH mixture.
Concrete implementation step is as follows:
The synthetic polypeptide of 5mg is added among the 7mg KLH, slowly add freshly prepared 3g/L glutaraldehyde solution 1ml, incubated at room 2h while shaking.Borate buffer dialysis 24h with pH8.5 obtains epitope 1-KLH mixture.
Embodiment 4: the preparation of polyvalent antibody
Get the epitope 1-KLH mixture of preparation among the 1-2mg embodiment 3, immune new zealand white rabbit, every 14 days booster immunizations once; Behind the 2nd booster immunization 7 days, ear vein was got blood, and separation of serum (the centrifugal 10min of 5000rpm) is collected supernatant, and survey is tired.Simultaneously according to identical step, with epitope 1 (SEQ ID NO:3) in contrast.
Concrete steps are as follows:
The epitope 1-KLH mixture of getting among the 1-2mg embodiment 3 preparation is fully emulsified with the complete freund adjuvant of equivalent, formation profit bag, and in the subcutaneous multi-point injection of rabbit neck part and back, every about 100 μ g.Booster immunization after 2 weeks, dosage is the same, after the full freund adjuvant that toos many or too much for use is fully emulsified, in the subcutaneous multi-point injection of rabbit back; Later on every 2 all booster immunizations 1 time; Since the 2nd booster immunization, each immunity was got tiring of hematometry antibody through ear vein after 7 days.
Wherein, to get the tire step of detection (ELISA method) of blood as follows for ear vein:
On 96 orifice plates, every hole adds 50 μ g/ml Tyrosine O-phosphates, 100 μ l, 4 ℃ spend the night after, wrap quilt, the washing.It is 1: 100 that the anti-Tyrosine O-phosphate immune serum of rabbit is diluted respectively, and 1: 500,1: 2500,1: 3200,1: 12800,1: 25600, every hole added 100 μ l, 37 ℃ of insulation 30min, washing.Each adds the goat-anti rabbit Ig enzyme enzyme conjugates 100 μ l of dilution in 1: 100,37 ℃ of insulation 30min, washing.Add TMB100 μ l, behind the 20min, add the H2SO4 termination reaction of 2mol/l.Use microplate reader to measure the A490nm value, be higher than the positive of 10 times of preimmune serums.
The above results meets the requirements, and satisfies in the rear neck artery bloodletting in 7 days of last booster immunization, collects blood sample.The blood sample of collecting was left standstill under 3-4 ℃ 3-4 hour, and 5000rpm is centrifugal 10 minutes then, collects serum, obtains polyvalent antibody (ear vein detects to tire and meets the requirements: KLH＞25600, behind naked peptide＞12800, the blood sample that carotid artery is got needn't detect again and tire).Aseptic subpackaged being stored in-80 is ℃ standby.
Embodiment 5: the preparation of anti-actin depolymerizing factor polyclonal antibody (how anti-)
The polyvalent antibody of preparation among the embodiment 5 is carried out purifying, make polyclonal antibody (how anti-).
With the Sepharose 4B coupling of epitope CKFKFQELKTRRG (SEQ ID NO:3) polypeptide and cyanogen bromide-activated, preparation polypeptide affinity column.
The polyvalent antibody of preparation is joined in the chromatography column of above preparation, be positioned over 4 ℃ of overnight incubation after, wash-out antibody namely obtains anti-actin depolymerizing factor polyclonal antibody (many anti-).
Embodiment 6: many anti-specificity checks at actin depolymerizing factor
Choose the tissue of paddy rice 93-11 different developmental phases and different sites, extract gross protein, with Bradford method working sample paddy rice total protein content, choose the tissue of paddy rice different developmental phases and different sites, extract gross protein, sample 10 μ g paddy rice total proteins on each sample, utilize the polyclonal antibody of preparation to detect protein expression, immunoblotting (Western blotting) can detect and the band of expecting that molecular weight (16kDa) is approaching, because target protein exists dimer and single aggressiveness, so target stripe is 32kD and 16Kd, with target protein 16KD be the relation of twice and a times, the back is verified this by experiment.This protein difference of expression amount all in the different tissues of paddy rice can further be studied the running balance of Actin muscle in paddy rice.
The preparation of gross protein sample: extremely Powdered with the above-mentioned fresh rice tissue of liquid nitrogen grinding, divide to install in the precooling centrifuge tube, per 300 μ l powder add 800 μ l protein cleavage liquid (62.5mmol/L pH7.4 TrisHCl, 10% glycerine, 2% SDS, 20mmol/LNaF, 2mmol/L EDTA, 1mmol/L PMSF, 5% beta-mercaptoethanol), rapid mixing also places on ice, hatches in the mixture of ice and water 10 minutes, and the concussion mixing was 1 time in per approximately 2 minutes.4 ℃, centrifugal 15 minutes of 12000r/min.Get supernatant, transfer in the new centrifuge tube ,-70 ℃ of preservations.Obtain the paddy rice total protein.
The Western marking:
The above-mentioned rice protein that extracts is carried out SDS-PAGE (12%), and electrotransfer is to pvdf membrane behind the SDS-PAGE, and the skim-milk with 5% seals pvdf membrane.Use prepare among the embodiment 7 to resist 1, incubated at room is 3 hours after diluting with 1: 1000 more, and (2mmol/LTrisHCl pH7.6,13.6mmol/L NaCl 0.1%Tween-20) wash film 3 times to TTBS, each 5 minutes.Add afterwards with the rabbit source of dilution in 1: 15000 how anti-(producer's article No.: middle China fir ZB2301), incubated at room 1 hour, TTBS washes film 3 times, each 5 minutes.Add ECL Pluc chromogenic reagent, darkroom exposure 5 minutes.The result as shown in Figure 1, a near specific band pvdf membrane is seen 16kDa, this is almost completely consistent with predicted molecular weight (16kDa).
Need to prove that although a lot of molecular weight albumen similar to it may be arranged in paddy rice whole protein, what be combined with how anti-specificity just has only one of actin depolymerizing factor.About specific prediction: SEQ ID NO:3 is (identical with the http://rapdb.dna.affrc.go.jp/ effect among the embodiment 1 through http://rice.plantbiology.msu.edu/blast.shtml, purpose is to check uniqueness again) inspection, determine that this peptide sequence is special, this sequence is unique definite actin depolymerizing factor in the paddy rice total protein.
In addition, in order further to prove the dimer of actin depolymerizing factor really and the actin depolymerizing factor of detected two bands, the total protein that above-mentioned 9 epi-positions are extracted respectively carries out the SDS-PAGE electrophoresis second time, obtain SDS glue, choose the band corresponding with the target stripe (16kDa) on the western blot then in the above, with beating mass spectrum after the Trypsin enzymic digestion, by the Syndicating search to one-level mass spectrum (Fig. 2) and second order ms figure (Fig. 3), can infer it is actin depolymerizing factor, the prediction of western blot confirms by experiment.
Although the specific embodiment of the present invention has obtained detailed description, it will be understood to those of skill in the art that.According to disclosed all instructions, can carry out various modifications and replacement to those details, these change all within protection scope of the present invention.Four corner of the present invention is provided by claims and any equivalent thereof.
Claims (16)
1. actin depolymerizing factor epitope, it is made up of the aminoacid sequence shown in the SEQ ID NO:3.
2. the composition for the preparation of anti-actin depolymerizing factor antibody wherein contains the described actin depolymerizing factor epitope of claim 1.
3. the composition of claim 2 is characterized in that, also contains the adjuvant that is useful on immunity.
4. actin depolymerizing factor epitope-carrier complexes, wherein, described actin depolymerizing factor epitope is the described actin depolymerizing factor epitope of claim 1, and described carrier is selected from keyhole limpet hemocyanin, BSA and casein.
5. the preparation method of an anti-actin depolymerizing factor antibody comprises that right to use requires the step of the mixture of the composition of 1 actin depolymerizing factor epitope or claim 2 or 3 or claim 4.
6. the preparation method of claim 5 is characterized in that, described anti-actin depolymerizing factor antibody is monoclonal antibody.
7. the preparation method of claim 5 is characterized in that described anti-actin depolymerizing factor antibody is polyclonal antibody.
8. the preparation method of claim 7, wherein, described preparation method comprises the steps:
1) blood sample that the mixture immune animal of the composition of the described actin depolymerizing factor epitope of claim 1 or claim 2 or 3 or claim 4 is obtained carries out centrifugally, obtains polyvalent antibody; With
2) purification step 1) in polyvalent antibody, obtain anti-actin depolymerizing factor polyclonal antibody.
9. polyvalent antibody, it is made by the actin depolymerizing factor epitope of claim 1 or the composition of claim 2 or 3 or the mixture immune animal of claim 4.
10. anti-actin depolymerizing factor antibody, it can be specifically in conjunction with the described actin depolymerizing factor epitope of claim 1.
11. the anti-actin depolymerizing factor antibody of claim 10, it is polyclonal antibody or monoclonal antibody.
12. the composition for detection of actin depolymerizing factor, it comprises claim 11 or 10 described anti-actin depolymerizing factor antibody.
13. an actin depolymerizing factor detection agent, it comprises claim 10 or 11 described anti-actin depolymerizing factor antibody.
14. claim 11 or the 10 described antibody purposes in the detection agent of preparation detection actin depolymerizing factor.
15. a method that detects actin depolymerizing factor, described method comprise that right to use requires the step of 10 or 11 described anti-actin depolymerizing factor antibody.
16. the described method of claim 15, wherein, described actin depolymerizing factor is the actin depolymerizing factor of paddy rice.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010520323 CN102206253B (en) | 2010-10-25 | 2010-10-25 | Actin depolymerizing factor epitope, actin depolymerizing factor antibody and use thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010520323 CN102206253B (en) | 2010-10-25 | 2010-10-25 | Actin depolymerizing factor epitope, actin depolymerizing factor antibody and use thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102206253A CN102206253A (en) | 2011-10-05 |
| CN102206253B true CN102206253B (en) | 2013-09-18 |
Family
ID=44695344
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201010520323 Active CN102206253B (en) | 2010-10-25 | 2010-10-25 | Actin depolymerizing factor epitope, actin depolymerizing factor antibody and use thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102206253B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102690787B (en) * | 2012-04-13 | 2013-03-13 | 湖南师范大学 | Hybridoma cells capable of secreting an anti human [beta]-actin protein monoclonal antibody and preparation method thereof |
| CN110655575B (en) * | 2019-10-14 | 2021-01-08 | 安徽医科大学 | Epitope polyclonal antibody of toxoplasma gondii Actin protein and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005184101A (en) * | 2003-12-16 | 2005-07-07 | Canon Inc | Image reader |
| WO2006012985A2 (en) * | 2004-07-28 | 2006-02-09 | Basf Plant Science Gmbh | Method for production of transgenic plants with increased pathogenic resistance by altering the content and/or activity of actin-depolymerising factors |
-
2010
- 2010-10-25 CN CN 201010520323 patent/CN102206253B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005184101A (en) * | 2003-12-16 | 2005-07-07 | Canon Inc | Image reader |
| WO2006012985A2 (en) * | 2004-07-28 | 2006-02-09 | Basf Plant Science Gmbh | Method for production of transgenic plants with increased pathogenic resistance by altering the content and/or activity of actin-depolymerising factors |
Non-Patent Citations (2)
| Title |
|---|
| BAC16183;T.Sasaki et al;《GenBank》;20080216;1-2 * |
| T.Sasaki et al.BAC16183.《GenBank》.2008,1-2. |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102206253A (en) | 2011-10-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Abd Alla et al. | Extracellular Domains of the Bradykinin B2 Receptor Involved in Ligand Binding and Agonist Sensing Defined by Anti-peptide Antibodies (∗) | |
| Atkinson et al. | The Anopheles-midgut APN1 structure reveals a new malaria transmission–blocking vaccine epitope | |
| Hou et al. | Development of peptide mimotopes of lipooligosaccharide from nontypeable Haemophilus influenzae as vaccine candidates | |
| Buch et al. | Brown spider (Loxosceles genus) venom toxins: Evaluation of biological conservation by immune cross-reactivity | |
| De Castro et al. | A Combined strategy to improve the development of a coral antivenom against Micrurus spp. | |
| White et al. | Crystal structure of a human MUC16 SEA domain reveals insight into the nature of the CA125 tumor marker | |
| Angeletti et al. | Plasmodium falciparum rosetting epitopes converge in the SD3-loop of PfEMP1-DBL1α | |
| CN102206253B (en) | Actin depolymerizing factor epitope, actin depolymerizing factor antibody and use thereof | |
| Ledesma et al. | A comparative analysis of the cross‐reactivity in the polcalcin family including Syr v 3, a new member from lilac pollen | |
| Molini et al. | B-cell epitope mapping of TprC and TprD variants of treponema pallidum subspecies informs vaccine development for human treponematoses | |
| Mittelman et al. | Monoclonal and polyclonal humoral immune response to EC HER‐2/neu peptides with low similarity to the host's proteome | |
| Yang et al. | Structural and functional characterization of Bc28. 1, major erythrocyte-binding protein from Babesia canis merozoite surface | |
| Atkinson et al. | Structural analysis of Anopheles midgut aminopeptidase N reveals a novel malaria transmission-blocking vaccine B-cell epitope | |
| CN103952383B (en) | Rice cytosolic type PPDK Protein Epitopes, its antibody and application | |
| Salgado-Mejias et al. | Structure of Rhoptry Neck Protein 2 is essential for the interaction in vitro with Apical Membrane Antigen 1 in Plasmodium vivax | |
| Nilsson et al. | Characterization of the Duffy‐Binding‐Like Domain of Plasmodium falciparum Blood‐Stage Antigen 332 | |
| CN102775486B (en) | The novel agent of monitoring injury of the kidney and test kit | |
| Alvarenga et al. | Design of antibody-reactive peptides from discontinuous parts of scorpion toxins | |
| CN110904059A (en) | Anthocyanin synthetase epitope peptide, antibody and application thereof | |
| Rajan et al. | Defining the complement C3 binding site and the antigenic region of Haemonchus contortus GAPDH | |
| CN102206254B (en) | Lysine decarboxylase epitope, anti-decarboxylase epitope antibody and application thereof | |
| Ledesma et al. | Olive pollen allergen Ole e 8: identification in mature pollen and presence of Ole e 8‐like proteins in different pollens | |
| CN102206251B (en) | Acyl carrier protein III epitope, acyl carrier protein III antibody and use thereof | |
| CN101955513A (en) | Beta-ureidopropionase epitope, anti-beta-ureidopropionase antibody and application thereof | |
| Shin et al. | IgE binding reactivity of peptide fragments of Bla g 4, a major German cockroach allergen |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: Epitopes of actin depolymerization factor, antibody against actin depolymerization factor and their applications Effective date of registration: 20200924 Granted publication date: 20130918 Pledgee: Qingdao West Coast Development (Group) Co.,Ltd.|Qingdao HAIC Group Financial Holding Co.,Ltd. Pledgor: BGI SHENZHEN Co.,Ltd. Registration number: Y2020440020012 |
|
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PC01 | Cancellation of the registration of the contract for pledge of patent right |
Granted publication date: 20130918 Pledgee: Qingdao West Coast Development (Group) Co.,Ltd.|Qingdao HAIC Group Financial Holding Co.,Ltd. Pledgor: BGI SHENZHEN Co.,Ltd. Registration number: Y2020440020012 |
|
| PC01 | Cancellation of the registration of the contract for pledge of patent right |