CN109870575A - A method of improving papaya ringspot virus antibody specificity - Google Patents
A method of improving papaya ringspot virus antibody specificity Download PDFInfo
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- CN109870575A CN109870575A CN201711257102.1A CN201711257102A CN109870575A CN 109870575 A CN109870575 A CN 109870575A CN 201711257102 A CN201711257102 A CN 201711257102A CN 109870575 A CN109870575 A CN 109870575A
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Abstract
The present invention discloses a kind of method for purifying papaya ringspot virus antibody with high specificity from antiserum, the filler surface that this method uses has used the polypeptide fragment of papaya ringspot virus coat protein different zones as aglucon, enables the specific antibody of filler absorbing shell albumen different zones.A kind of purification process carries out the purifying of antibody using above-mentioned filler.
Description
Technical field
The present invention relates to the methods for purifying papaya ringspot virus antibody with high specificity from antiserum, belong to albumen
Separation technology field and technical field of immunological detection.
Background technique
Papaya ringspot virus is the main virus of main harm papaya, can result in papaya occur floral leaf, ring spot,
The symptoms such as deformed fruit and plant early ageing, have seriously affected papaya production.
In recent years, people go deep into research work, and PRSV is infected in discovery, and there are a large amount of strains, the change of isolate
Different, these variations lead to different strains pathogenic, and there are bigger differences, wherein strong pathogenic strain can result in more serious illness,
And low virus influence orchid degree is lower.Furthermore with further going deep into for research, it has also been found that the metainfective orchid of weak diseased plant
Flower can slow down the infection harm of strong diseased plant, and there are a degree of cross-protections;On the other hand, people are disease-resistant in popularization
Found when transgenic papaya, transgenic papaya to the resistance of different virus strain there are apparent difference, it is especially homologous
The low strain of property does not have resistance.It can be seen that precise Identification virus and virus strain are most important for preventing and controlling.
The detection of the virus mainly uses the serological techniques such as indirect ELISA and double crush syndrome at present, these technologies
Accuracy depend on detection antibody (IgG) specificity.Detection antibody main source is immune by the way that virus to be used as
Original annotation is mapped to the mammals such as rabbit, sheep, mouse, to induce the antibody that animal generates specific bond virus.Further collect
Animal blood serum just contain a large amount of antiviral antibody, can be used for general detection work.But, special to further increase antibody
Property, it is therefore necessary to the concentration of antibody is further increased by antibody purification procedures and reduces the content of non-antibody protein.
Can be used in the technology of antibody purification at present includes: salting out method, caprylic acid precipition, ion-exchange and affinity chromatography
Method etc..The most commonly used is affinity chromatography, this method is the specific recognition knot using receptor and ligand, antibody and antigen
Enrichment target product is closed, its advantage is that can be by simply handling to obtain the higher target product of purity.The affine layer of antibody
Analysis can be used Protein A, Protein G, antiantibody, antigen etc. as aglucon and prepare affine filler, most commonly adopt
Albumin A or Protein G is used to prepare affine filler as aglucon.Wherein albumin A is a kind of albumen on Staphylococcus aureus cell wall
Matter, can be specifically in conjunction with the area Fc of mammalian antibody (mainly IgG), and Protein G is Streptococcal cells wall-held protein, energy
Enough in conjunction with the constant region of IgG antibody, albumin, alpha2-macroglobulin.Therefore, it is purified using the affine filler of albumin A, Protein G
Obtained antibody belongs to mixed antibody, and the specificity of this antibody is poor.
Typical method using albumin A, protein g affinity chromatography antibody purification is as follows: (1) being filled out by functional reagent modification
Expect that surface forms NH2 or COOH group, NH2 or COOH is further modified by glutaraldehyde or bifunctional group obtains activation and fill out
Material;(2) albumin A of purifying or Protein G are added in Activation filling, by being covalently bound to filler, affine fill out is prepared
Material;(3) make filler of the thick solution of antiserum Jing Guo fixed protein A goods Protein G, be adsorbed onto IgG on filler;(4) with neutrality
Buffer rinses filler, removes other impurities;(4) it is adsorbed on filler using acidic buffer (pH2.5 ~ pH3.0) elution
IgG;(5) adjustment eluent pH value is to neutrality, the IgG purified.
Furthermore it is also possible to prepare affine filler as aglucon using antigen, it is applied to the available affinity of antibody purification
Good, high specificity, purity is high antibody.(2015103209206) are invented by the denatured antigen albumen (inclusion body of such as prokaryotic expression
Albumen) it is coupled at solid phase carrier directly with antibody purification, operating procedure is simplified, that is, prepares soluble antigen protein process, institute
Obtaining antibody has high purity and specificity, is suitable for applying in laboratory preparation and industrial production.Document seals the swine fevers such as beautiful jade
The Sepharose-4B of viral antigen and Epichlorohydrin activation coupling is prepared into immune affinity chromatographic column for the antiviral antibody
Purifying, obtain purity is high, the strong antiviral antibody of activity.But, the antibody identification due to purifying using holoantigen
Mixed antibody is also belonged to, the precise Identification of pathogen strain, isolate is not used to.
In consideration of it, can be used in a series of polypeptides of purification specificity antibody from antiserum the present invention provides a kind of and match
Base affinity media meets the needs of antibody purification.
Summary of the invention
The purpose of the present invention is utilizing PRSV CP amino acid sequence information, a series of polypeptide fragments are synthesized as aglucon idol
It is linked to filler and prepares a series of affine filler, substitute conventional affine filler, obtain needle for purifying from PRSV antiserum
To the antibody of different fragments.This method can purify to obtain the detection antibody for viral particular amino acid residue, specificity
It is and easy to operate, simple to equipment requirement much higher than the antibody that albumin A, protein g affinity chromatography purify.
In order to realize the purpose, the technical scheme is that according to PRSV CP amino acid sequence, preferably have certain
Hydrophily, surface accessibility or antigenic sequence, design length are a series of polypeptide fragments of 6-30 amino acid residue;Using
Amino silane is to including cellular glass, glass fibre, silica gel, resin, Ago-Gel, magnetic particle material, cellulose (such as cotton
Flower, fiber chromatographic paper) carry out amination modify to obtain amido modified filler;It is repaired using conventional Fmoc or Boc method in above-mentioned amino
Decorations filler further synthesizes linking arm " GnS " (wherein n represents the quantity of G, respectively 3-20), and end has NH2 group;With
Above-mentioned surface modification filler or other common fillers with NH2 group are substrate, carry out polypeptide using Fmoc method or Boc method
The fabricated in situ of segment obtains the affine filler of polypeptide aglucon;The present invention also includes obtaining polypeptide fragment using conventional chemical synthesis
It afterwards, will by glutaraldehyde method, active ester method, carbodlimide method, succinic anhydride method and diazotising method or other common coupling methods
Polypeptide fragment is coupled to filler and affine filler is prepared.According to polypeptide and the lower characteristic of affinity of antibody, in antibody purification
In the process, the preferred elution buffer of pH3.5-4.5, to reduce to the active destruction of antibody.
Experimental procedure by the invention is
1) design of polypeptide fragment: first according to PRSV CP conserved amino acid sequences, selection is located at the piece of the special conserved region of virus
Section;Further analyze the hydrophily, surface accessibility and antigenicity of these fragment amino acid residues;Preferably include CP albumen n end
To C-terminal, most it is short be 6 amino acid lengths, up to 30 amino acid lengths a series of polypeptide fragments.
2) filler is amido modified: using 0.1%-20% amino silane solution, is added in filler in being stored at room temperature 30
Min-12 h carries out the NH2 modification of filler.The enterprising one-step synthesis connection of filler modified using conventional Fmoc or Boc method in NH2
Arm " GnS ".
3) fabricated in situ of polypeptide fragment: using Fmoc method or Boc method above-mentioned surface with amido modified filler or
Other have carries out fabricated in situ polypeptide on NH2 group filler, the polypeptide is above-mentioned preferred polypeptide.
4) it the fixation of polypeptide fragment aglucon: after the synthesis for carrying out above-mentioned preferred polypeptide fragment using chemical synthesis, adopts
With glutaraldehyde method, active ester method, carbodlimide method, succinic anhydride method and diazotising method or other common coupling methods by polypeptide piece
Section, which is coupled to filler equally, can be prepared into identical affine filler.
5) it the purifying of antibody: after antiserum is diluted with 0.05M-0.2M PBS, is added to prepared by the present invention a series of
1-3 hours are stood in affine filler, then washs removal nonspecific antibody and other impurities with PBS solution;PH3.0-4.0 is used again
Glycine-HCI buffer, citrate buffer solution or it is other elution solution carry out antibody elution.The system that will be eluted
Column antibody-solutions NaOH, NaCO3Equal alkaline solutions adjust pH to neutrality, obtain a series of antibody of purifying after desalination of dialysing.
Main advantages of the present invention be by purifying a series of available antibody for the different protein fragments of virus, and
And it can ensure that the activity of antibody using relatively mild elution requirement.
Embodiment one
For a better understanding of the present invention, this embodiment be PRSV CP "1SKNETADAGL10" for polypeptide fragment, description is directed to
The preparation method of the antibody of the segment is affine filler;And further compare the spy of antibody purification and albumin A affinity purification antibody
The advantages of opposite sex is tested, and illustrates affine filler of the invention.
Method used in following embodiments is conventional method unless otherwise specified.
Material used in following embodiments, reagent etc., are commercially available unless otherwise specified.
1 material
1.1 major experimental equipment and consumptive material
Glass fibre is market purchase;Using 3 intramuscular injection and 1 intravenous immune program, exempt from by immunogene of PRSV
Epidemic disease rabbit, measurement potency acquire rabbit anti-serum after reaching 1:5000.
2 methods
The surface NH2 of 2.1 glass fibres is modified: being taken 10 g glass fibres, is impregnated 5 with the concentrated sulfuric acid and 30% hydrogen peroxide (v/v) 3:1
Min is cleaned with pure water to neutrality, 120 DEG C of heated-air dryings.After glass fibre is cooled to room temperature, be added 5% amino silane without
Hydrous ethanol solution is impregnated 4 hours, and after being filtered dry, with 10 times of volume pure water rinsings, 80 DEG C of heated-air dryings obtain amido modified glass
Glass fiberfill.
The fabricated in situ of 2.2 polypeptide fragments: using Fmoc method based on the NH2 group of filler surface, synthesizes 1 first
The serine of equivalent further synthesizes 3 continuous Formation of glycine " GGGS " polypeptide linking arms, is finally synthesizing PRSV CP
The corresponding polypeptide fragment of 171-179aa " SKNETADAGL ", the affinity chromatography for forming " SKNETADAGLGGGS- glass fibre " are situated between
Matter.It is condensing agent that amino and carboxyl condensation reaction of the present invention, which uses HBTU, DIEPC, is carried out using conventional solid synthetic method.
2.3 antibody purifications: affine filler is added in 50 mL affinity columns, balances standby with 100 mL, 0.1 M PBS
With;It is added in affinity column after diluting 1 mL antiserum using 20 mL PBS, 37 DEG C of incubations, 1 hour absorption specificity antibody;Make
Removal non-specific antibody and impurity are eluted with 100 mL PBS;After reusing the elution of 0.005 M phosphoric acid solution, with 2 times of volumes
0.005 NaOH is neutralized to neutrality, finally obtains 1 mL of antibody purification using PEG8000 dehydration and semi-permeable membrane dialysis.
The regeneration of 2.4 affinity columns: affine filler is rinsed, again with pure water rinsing into 10 times of volume 0.01M phosphoric acid
Property, the PBS with 5 times of volumes containing 20% ethyl alcohol balances affinity column, is stored in 4 DEG C.
2.5 antibody purification concentration mensurations: using BSA as standard, drawing standard curve using Coomassie brilliant G-250 method, into
Row antibody purification concentration mensuration.
The measurement of 2.6 antibody titers: with the virion coated elisa plate of 1 ng/mL, and being coated with BSA is negative control, is adopted
Antibody titer is measured respectively with antiserum before purification and purified antibodies.
The experiment of 2.7 antibody specificities
Detected respectively using antiserum before purification and purified antibodies the papaya blade of infection PRSV, healthy papaya blade,
The samples such as marmor upsilon positive control and healthy potato leaf, compare influence of the purification experiment to antibody specificity.
3 results
3.1 antibody purification concentration: Coomassie brilliant G-250 method is used to measure the concentration for showing antibody purification for 4 ng/mL, about
It is the 1/800 of antiserum concentration, shows that purification experiment eliminates a large amount of non-specific protein.
The measurement of 3.2 antibody titers: the potency of antiserum and antibody purification before purification is measured by indirect ELISA, as a result table
Bright antiserum titre before purification is 1:5000;Antibody titer 1:2000 after purification.
The experiment of 3.3 antibody specificities: there are apparent compatible reaction, colour developings for antiserum and PRSV positive sample before purification
Significant reaction (A 405=0.427 ± 0.021), but for there is certain intersection when detecting marmor upsilon positive sample
React (A405=0.278 ± 0.001), and detect healthy sample colour developing colour developing background it is higher (A 405=0.174 ± 0.012), shadow
The evaluation of testing result is rung;Obvious compatible reaction occurs for antibody and PRSV positive sample after purification, although chromogenic reaction has
Lower (A 405=0.352 ± 0.004), but occur with healthy papaya blade cross reaction (A 405=0.034±
0.017), also with marmor upsilon positive control occur cross reaction (A 405=0.041 ± 0.011).
Embodiment two
For a better understanding of the present invention, this embodiment be PRSV CP "29KDKDNDGASDGNDVST44" for polypeptide fragment,
Preparation method of the description for the affine filler of antibody of the segment;And further compare antibody purification and albumin A affinity purification
The specificity experiments of antibody, the advantages of illustrating affine filler of the invention.
Method used in following embodiments is conventional method unless otherwise specified.
Material used in following embodiments, reagent etc., are commercially available unless otherwise specified.
1 material
1.1 major experimental equipment and consumptive material
100 mesh silica gel are market purchase;Using 3 intramuscular injection and 1 intravenous immune program, exempt from by immunogene of PRSV
Epidemic disease rabbit, measurement potency acquire rabbit anti-serum after reaching 1:5000.
2 methods
The surface NH2 of 2.1 glass fibres is modified: being taken 10 g, 100 mesh silica gel, is impregnated with the concentrated sulfuric acid and 30% hydrogen peroxide v/v3:1
5 min are cleaned with pure water to neutrality, 120 DEG C of heated-air dryings.After silica gel is cooled to room temperature, it is anhydrous that 5% amino silane is added
Alcohol solution dipping 4 hours, after being filtered dry, with 10 times of volume pure water rinsings, 80 DEG C of heated-air dryings obtained amido modified silica gel
Filler.
The fabricated in situ of 2.2 linking arms: using Fmoc method based on the NH2 group of filler surface, synthesizes 1 first and works as
The serine of amount, further synthesizes 3 continuous Formation of glycine " GGGS " polypeptide linking arms, and end has NH2 group.
2.3 obtain the polypeptide fragment " KDKDNDGASDGNDVST " of 10 mg using chemical synthesis, pass through glutaraldehyde method idol
It is linked to filler linking arm NH2, obtains the affinity chromatography medium of " KDKDNDGASDGNDVSTGGGS- silica gel ".
2.4 antibody purifications: affine filler is added in 50 mL affinity columns, balances standby with 100 mL, 0.1 M PBS
With;It is added in affinity column after diluting 1 mL antiserum using 20 mL PBS, 37 DEG C of incubations, 1 hour absorption specificity antibody;Make
Removal non-specific antibody and impurity are eluted with 100 mL PBS;After reusing the elution of 0.005 M phosphoric acid solution, with 2 times of volumes
0.005 NaOH is neutralized to neutrality, finally obtains 1 mL of antibody purification using PEG8000 dehydration and semi-permeable membrane dialysis.
The regeneration of 2.4 affinity columns: affine filler is rinsed, again with pure water rinsing into 10 times of volume 0.01M phosphoric acid
Property, the PBS with 5 times of volumes containing 20% ethyl alcohol balances affinity column, is stored in 4 DEG C.
2.5 antibody purification concentration mensurations: using BSA as standard, drawing standard curve using Coomassie brilliant G-250 method, into
Row antibody purification concentration mensuration.
The measurement of 2.6 antibody titers: with the virion coated elisa plate of 1 ng/mL, and being coated with BSA is negative control, is adopted
Antibody titer is measured respectively with antiserum before purification and purified antibodies.
The experiment of 2.7 antibody specificities: it is detected respectively using Protein A purification antibody and the affine filler antibody purification of the present invention
Infect the samples such as papaya blade, healthy papaya blade, marmor upsilon positive control and the healthy potato leaf of PRSV
Product compare influence of the purification experiment to antibody specificity.
3 results
3.1 antibody purification concentration: Coomassie brilliant G-250 method is used to measure the concentration for showing antibody purification for 7 ng/mL, about
It is the 1/500 of antiserum concentration, shows that purification experiment eliminates a large amount of non-specific protein.
The measurement of 3.2 antibody titers: the potency of antiserum and antibody purification before purification is measured by indirect ELISA, as a result table
Bright antiserum titre before purification is 1:5000;Antibody titer 1:1000 after purification.
The experiment of 3.3 antibody specificities: there are apparent compatible reaction, colour developings for Protein A purification antibody and PRSV positive sample
Significant reaction (A 405=0.514 ± 0.01), but anti-for there is certain intersection when detecting marmor upsilon positive sample
Answer (A 405=0.297 ± 0.014), and detection healthy sample colour developing colour developing background it is higher (A 405=0.177 ± 0.004) it, influences
The evaluation of testing result;Obvious compatible reaction occurs for antibody and PRSV positive sample after purification, although chromogenic reaction is
Lower (A 405=0.447 ± 0.011), but occur with healthy papaya blade cross reaction (A 405=0.051 ± 0.005),
Also not with marmor upsilon positive control occur cross reaction (A 405=0.021 ± 0.001).
Although causing potency to decline, obtained antibody specificity it can be seen that the antibody yield that purifying obtains is lower
Significantly risen, is more advantageous to the accuracy for improving detection.
Claims (4)
1. a kind of method for improving papaya ringspot virus (PRSV) antibody specificity, it is characterised in that: modified in filler surface
The polypeptide fragment of PRSV coat protein different zones makes it have the feature for capableing of the antibody of absorbing shell albumen different zones,
Conventional affinity chromatography filler is substituted, antibody purification is carried out.
2. polypeptide fragment according to claim 1, it is characterised in that: the polypeptide fragment corresponds to PRSV coat protein
Amino acid sequence.
3. polypeptide fragment according to claim 1, it is characterised in that: the polypeptide fragment length is 6-30 amino acid
Residue.
4. described according to claim 1, the method for modifying of filler, it is characterised in that: connected using Fmoc method or Boc method in polypeptide
Connect the enterprising one-step synthesis polypeptide aglucon of arm;Or using after chemically synthesized polypeptide segment, filler is coupled to by albumen coupling method
In.
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Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103412121A (en) * | 2013-07-20 | 2013-11-27 | 福建农林大学 | Colloidal gold immune test strip for rapid detection of Papaya ringspot virus |
CN107091928A (en) * | 2017-06-30 | 2017-08-25 | 厦门华侨亚热带植物引种园 | A kind of affine filler of cymbidium mosaic virus antibody and preparation method thereof |
-
2017
- 2017-12-04 CN CN201711257102.1A patent/CN109870575A/en active Pending
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103412121A (en) * | 2013-07-20 | 2013-11-27 | 福建农林大学 | Colloidal gold immune test strip for rapid detection of Papaya ringspot virus |
CN107091928A (en) * | 2017-06-30 | 2017-08-25 | 厦门华侨亚热带植物引种园 | A kind of affine filler of cymbidium mosaic virus antibody and preparation method thereof |
Non-Patent Citations (2)
Title |
---|
ROHINI BHAT VALEKUNJA,ET AL: "The detection of papaya ringspot virus coat protein using an electrochemical immunosensor", 《ANAL. METHODS》 * |
赵芹 等: "番木瓜环斑病毒外壳蛋白基因原核表达蛋白的抗血清制备及其检测应用", 《园艺学报》 * |
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