CN109897090A - A kind of brown paddy plant hopper Vg polypeptide and its how anti-preparation method - Google Patents
A kind of brown paddy plant hopper Vg polypeptide and its how anti-preparation method Download PDFInfo
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- CN109897090A CN109897090A CN201811579110.2A CN201811579110A CN109897090A CN 109897090 A CN109897090 A CN 109897090A CN 201811579110 A CN201811579110 A CN 201811579110A CN 109897090 A CN109897090 A CN 109897090A
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Abstract
The invention discloses the preparation methods of a kind of brown paddy plant hopper Vg polypeptide and its polyclonal antibody.The amino acid sequence of the Vg polypeptide are as follows: NPASNSESNQRSSH.Its Anti-TNF-α preparation step is as follows: (1) brown paddy plant hopper Vg Protein Epitopes are analyzed；(2) brown paddy plant hopper Vg polypeptide design and synthesis；(3) synthesis polypeptide and carrier protein are crosslinked；(4) rabbit-anti brown paddy plant hopper Vg polypeptide antibody is prepared；(5) it collects, isolated serum antibody-containing, antibody purification is to get the antibody for arriving brown planthopper resistant Vg polypeptide.The brown paddy plant hopper Vg polypeptide polyclonal antibody specificity that the present invention obtains is good, with high purity, and specific binding hair can occur with brown paddy plant hopper Vg albumen and answer, can be used for the correlative study of brown paddy plant hopper Vg albumen, such as the analysis of its characteristic, function, express spectra and content.
Description
Technical field
The present invention relates to a kind of polypeptide and its preparation method for antibody, this antibody is mainly used for the inspection of native protein antigen
It surveys, the preparation method of specifically a kind of rabbit-anti brown paddy plant hopper Vg polypeptide polyclonal antibody.
Background technique
Vg full name is vitellogenin, is vitellogenin, is played extremely in the reproductive processes such as insect ovum generation
Close important role.Brown paddy plant hopper is agriculture important pests, the Vg of brown paddy plant hopper is studied or detected the prevention and treatment for brown paddy plant hopper
It is of great significance.
Summary of the invention
(1) the purpose of the present invention is to provide a kind of Vg polypeptide, sequences are as follows: NPASNSESNQRSSH.
(2) that another object of the present invention is to provide a species specificity is good, with high purity, can be with natural Vg in tissue or cell
Vg polypeptide polyclonal antibody of albumen specific recognition and preparation method thereof.
(3) the Vg polypeptide polyclonal antibody described in is achieved through the following technical solutions:
Step 1: when synthesizing the polypeptide sequence, increase a cysteine in its N-terminal, convenient and carrier protein couplet is used
Polypeptide automatic synthesizer synthesizes Vg modified polypeptide, after purification with carrier protein KLH, forms the compound egg of Vg modified polypeptide-KLH
It is white；
Step 2: the Vg modified polypeptide-KLH compound protein after emulsification is subcutaneously injected in rabbit back, after initial immunity, carries out 3
Secondary booster immunization；
Step 3: it collects, the isolated serum containing rabbit-anti brown paddy plant hopper Vg modified polypeptide antibody；
Step 4: antiserum is subjected to peptide affinity purification by Vg polypeptide affinity column, obtains Vg polypeptide antibody；
Step 5: bioactivity is carried out to Vg polypeptide antibody；
Step 6: it is identified by specificity of the Western Blot to Vg polypeptide antibody.
Detailed description of the invention
Fig. 1 is Western Blot figure, and the purpose band size of immunoblotting is in 200kDa or so in figure, with Vg albumen
Theoretical molecular weight (227.8kDa) is in the same size.
Specific embodiment
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are only used for explaining this hair
It is bright, rather than limit the scope of the invention.Under the premise of without departing substantially from technical solution of the present invention, made for the present invention
Field those of ordinary skill any change easy to accomplish is fallen within scope of the presently claimed invention.
Embodiment one: the analysis and the design and synthesis of Vg polypeptide of brown paddy plant hopper Vg sequence
According to the brown paddy plant hopper Vg sequence (accession number: AEL22916) on GenBank, it is known that brown paddy plant hopper Vg protein sequence includes 2045
A amino acid analyzes brown paddy plant hopper Vg protein characteristic using the Protean program module in DNAstar software, learns this
Molecular weight of albumen is 227786.41 dalton, and isoelectric point 8.32 is basic protein, further analyzes the Argine Monohydrochloride sequence
The features such as antigenicity, hydrophily, the surface possibility of column therefrom screen the polypeptide sequence that one section of sequence is NPASNSESNQRSSH
Column are suitable as antigen (339aa-352aa).To increase the immunogenicity of polypeptide, above-mentioned more convenient for being crosslinked with carrier protein
The C-terminal of peptide increases a cysteine C, therefore final polypeptide sequence to be synthesized is NPASNSESNQRSSHC.Peptide systhesis by
The polypeptide of the synthesis of polypeptide automatic synthesizer, synthesis detects purity with high performance liquid chromatograph (HPLC), and purity 87.2% uses MS
Mass spectrograph detects the molecular weight of polypeptide, molecular weight 1617.40.
Embodiment two: polypeptide and carrier protein are crosslinked
Carrier protein KLH is crosslinked as crosslinking agent with synthesis polypeptide using MBS: dissolving KLH to dense with cross-linking buffer
Degree is 10mg/mL；Dissolution MBS is 10mg/mL in DMF；By dissolved KLH solution and MBS solution in 10:1(W/W) ratio
It mixes, room temperature activates KLH 30 minutes；With Sephadex G-25 purify activation KLH solution；By the KLH solution of activation and more
Peptide solution presses 1:1(W/W) mixing, it reacts at room temperature 3 hours；By above-mentioned reaction solution in PBS dialysed overnight at 4 DEG C, obtain polypeptide-
KLH compound protein.
Embodiment three: experimental animal is immunized
Of the right age new zealand male rabbit is chosen as immune animal, ear venous blood collection 2-3mL before being immunized is used as subsequent ELISA and examines
The negative control of survey.When first immunisation, the polypeptide-K LH compound protein of 0.5mg is dissolved in the PBS solution of 0.5mL, with equal bodies
Long-pending Freund's complete adjuvant mixes well emulsification, multi-point injection under rabbit skin.After 2 weeks, booster immunization for the first time is carried out, by 0.5mg
Polypeptide-K LH crosslinking compound protein be dissolved in the PBS solution of 0.5mL, mix well cream with isometric incomplete Freund's adjuvant
Change, subcutaneous multi-point injection, the booster immunization hereafter equally operated every 3 weeks, front and back totally 3 times.Each booster immunization 1 week
Afterwards, from rabbit ear, venous blood collection is micro, with indirect elisa method detect immune serum potency, when potency reach 1:20000 with
On, rabbit blood is collected by the way of arteria carotis bloodletting, prepares serum.
Example IV: Vg polypeptide affinity column antibody purification
The gel suspension of 1mL activation is injected into chromatographic column, after dried liquid stream in column, the coupling buffer that 5mL is added rinses layer
Analyse column.The Vg polypeptide synthesized with the dissolution of 1mL coupling buffer, and chromatographic column is added, the coupling buffer for adding 1mL extremely chromatographs
In column, mixing overnight is rotated under the conditions of 4 DEG C.Chromatographic column is rinsed with 8mL coupling buffer, then is sealed with the confining liquid room temperature of 3mL
It closes 1 hour, is rinsed chromatographic column 3 times with combination buffer, until dried liquid stream in column, is prepared Vg polypeptide combination chromatographic column.
The combination buffer containing Vg antibody serum is added into chromatographic column, room temperature mixes 1 hour, rinses layer with 30mL dcq buffer liquid
Column is analysed, until the 280nm absorption peak of efflux is stablized.With the elution buffer elution chromatography column of 15mL, the Vg for obtaining purifying is anti-
Body.
Embodiment five: the potency of indirect elisa method measurement antibody
Vg modified polypeptide-KLH compound is coated with elisa plate, is coated with overnight at 4 DEG C；It is small with 5% skimmed milk power room temperature closing 2
When；Make various concentration dilution with Vg antibody, 1:1000,1:2000,1:4000,1:8000,1:16000,1:32000,1:
64000,1:128000,1:256000,1:512000 are incubated overnight for room temperature 2 hours or 4 DEG C；Horseradish peroxidase label is added
Goat-anti rabbit secondary antibody, be incubated at room temperature 2 hours；TMB is added and carries out chromogenic reaction, sulfuric acid measures 450nm wavelength after terminating reaction
Absorbance.When being greater than 2.1 with the ratio of negative serum, calculating antibody potency.It is detected, the effect of the Vg antibody after purifying
Valence reaches 1:512000.
The specific detection of the analysis Vg antibody of embodiment six: WesternBlot
SDS-PAGE gel is prepared according to standard method, the Tissue lysates that 10 μ L protein concentrations are 5mg/ml are added
In the loading hole of Vertial electrophorestic tank, constant pressure 80V electrophoresis ran concentration glue to sample, when being in straight line, changed 120V voltage,
Electrophoresis terminates electrophoresis when running out of separation gel completely to bromophenol blue indicator, is turned 90 minutes using wet transferring film method constant pressure 100V electricity,
By albumen transferring film to NC film.The Vg antibody that example IV is obtained is as primary antibody, after 1:500 dilution, with the NC after above-mentioned wet turn
Film hybridizes 2 hours at room temperature, is then hybridized at room temperature 2 hours with the goat anti-rabbit antibody that HRP is marked, using DAB development process
It develops the color, obtains immunoblot results.The present embodiment the experimental results showed that, Vg is generally expressed in brown paddy plant hopper ovary.
Sequence table
<110>China's metering university
<120>a kind of brown paddy plant hopper Vg polypeptide and its how anti-preparation method
<130> 2018
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 14
<212> PRT
<213>brown paddy plant hopper (Nilaparvata lugens)
<400> 1
Asn Pro Ala Ser Asn Ser Glu Ser Asn Gln Arg Ser Ser His
1 5 10
Claims (5)
1. a kind of Vg polypeptide, it is characterised in that: amino acid sequence NPASNSESNQRSSH.
2. a kind of preparation method for antibody of rabbit-anti brown paddy plant hopper Vg polypeptide, it is characterised in that: by Vg polypeptide sequence described in claim 1
The modified polypeptide of synthesis and carrier protein are cross-linked to form compound protein, animal are immunized with compound protein, takes by synthetic modification polypeptide
The blood of immune animal prepares antiserum, and antibody is isolated and purified from serum.
3. a kind of preparation method for antibody of rabbit-anti brown paddy plant hopper Vg polypeptide according to claim 2, it is characterised in that: described to repair
Adoring polypeptide is amino acid sequence tail end one cysteine residues of increase in claim 1, and the carrier protein is keyhole blood
The amino of azurin (KLH) or bovine serum albumin(BSA) (BSA), the sulfydryl of the modified polypeptide and the carrier protein is by being crosslinked
Agent covalent cross-linking forms compound protein.
4. a kind of preparation method for antibody of rabbit-anti brown paddy plant hopper Vg polypeptide according to claim 3, it is characterised in that: described multiple
After hop protein and immunologic adjuvant mixing and emulsifying, in rabbit back by being subcutaneously injected, after initial immunity, by 3 booster immunizations, obtain
To antiserum.
5. a kind of preparation method for antibody of rabbit-anti brown paddy plant hopper Vg polypeptide according to claim 4, it is characterised in that: right is wanted
Antiserum obtained in asking 4 can obtain the antibody of high-purity after polypeptide affinity purification from antiserum.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023010682A1 (en) * | 2021-08-05 | 2023-02-09 | 广东省农业科学院作物研究所 | Antibody capable of detecting nicotiana tabacum agglutinin protein, preparation method therefor and application thereof |
| CN117106058A (en) * | 2023-08-29 | 2023-11-24 | 扬州大学 | Preparation method and application of pig neuromedin B receptor polypeptide and polyclonal antibody thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104861059A (en) * | 2015-04-29 | 2015-08-26 | 中国计量学院 | Brown planthopper VgR polypeptide and multi-resistant preparation method thereof |
| CN104974245A (en) * | 2015-04-29 | 2015-10-14 | 中国计量学院 | Brown planthopper VgR polypeptide and brown planthopper VgR polypeptide polyclonal antibody preparation method |
-
2018
- 2018-12-24 CN CN201811579110.2A patent/CN109897090B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104861059A (en) * | 2015-04-29 | 2015-08-26 | 中国计量学院 | Brown planthopper VgR polypeptide and multi-resistant preparation method thereof |
| CN104974245A (en) * | 2015-04-29 | 2015-10-14 | 中国计量学院 | Brown planthopper VgR polypeptide and brown planthopper VgR polypeptide polyclonal antibody preparation method |
Non-Patent Citations (3)
| Title |
|---|
| DONG,X.等: "GenBank: AEL22916.1", 《GENBANK》 * |
| DOR-JIH等: "Determination and distribution of a femaleispecific protein in the brown planthopper, Nilaparvata lugens Stal (Homoptera: Delphacidae)", 《TISSUE AND CELL》 * |
| 俞叶微等: "褐飞虱吞蛋白的基因克隆、多克隆抗体制备及表达定位", 《昆虫学报》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023010682A1 (en) * | 2021-08-05 | 2023-02-09 | 广东省农业科学院作物研究所 | Antibody capable of detecting nicotiana tabacum agglutinin protein, preparation method therefor and application thereof |
| CN117106058A (en) * | 2023-08-29 | 2023-11-24 | 扬州大学 | Preparation method and application of pig neuromedin B receptor polypeptide and polyclonal antibody thereof |
| CN117106058B (en) * | 2023-08-29 | 2024-05-14 | 扬州大学 | Preparation method and application of pig neuromedin B receptor polypeptide and polyclonal antibody thereof |
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| CN109897090B (en) | 2023-01-10 |
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