CN107091928A - A kind of affine filler of cymbidium mosaic virus antibody and preparation method thereof - Google Patents
A kind of affine filler of cymbidium mosaic virus antibody and preparation method thereof Download PDFInfo
- Publication number
- CN107091928A CN107091928A CN201710526035.2A CN201710526035A CN107091928A CN 107091928 A CN107091928 A CN 107091928A CN 201710526035 A CN201710526035 A CN 201710526035A CN 107091928 A CN107091928 A CN 107091928A
- Authority
- CN
- China
- Prior art keywords
- antibody
- filler
- preparation
- affine
- mosaic virus
- 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.)
- Pending
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Virology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Molecular Biology (AREA)
- Hematology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention discloses a kind of affine filler of cymbidium mosaic virus antibody and preparation method thereof, filler surface is modified using amino silane, after fabricated in situ linking arm, the aglucon further modified is the polypeptide fragment of cymbidium mosaic virus coat protein different zones, enables the specific antibody of filler absorbing shell albumen different zones.A kind of affine filler of cymbidium mosaic virus antibody its prepared by the preparation method of above-mentioned affine filler.The affine filler of the present invention can be used in detection antibody of the purifying for coat protein different zones, can be used for Viral diagnosis work, can be used for virus strain, the difference appraisal of isolate.
Description
Technical field
The present invention relates to a series of affine fillers for purifying cymbidium mosaic virus antibody with high specificity from antiserum
And preparation method thereof, belong to protein separation technology field and technical field of immunological detection.
Background technology
Cymbidium mosaic virus is one kind virus of main harm orchid, and can result in orchid, floral leaf, ring spot, flower occur abnormal
The symptom such as shape and plant early ageing, has had a strong impact on the ornamental values and the economic values of orchid, and inspection is classified as by many countries and regions
Epidemic disease object.The current viral detection is mainly using serological techniques, these technologies such as indirect ELISA and double crush syndromes
Accuracy depend on detection antibody (IgG) specificity.
Detection antibody main source be by using virus as immunogen injection to mammals such as rabbit, sheep, mouse, so as to lure
Thing is started to produce the antibody of specific bond virus.The animal blood serum further collected just contains substantial amounts of antiviral antibody, can
For general detection work.But, it is further raising antibody specificity, it is therefore necessary to further by antibody purification procedures
Improve the concentration of antibody and the content of reduction 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..Most common of which is affinity chromatography, and this method is the specific recognition knot with part, antibody and antigen using acceptor
Enrichment target product is closed, its advantage can be by simple processing and obtain the higher target product of purity.The affine layer of antibody
Analysis can use Protein A, Protein G, antiantibody, antigen etc. as aglucon and prepare affine filler, most commonly adopt
With albumin A or Protein G affine filler is prepared as aglucon.Wherein albumin A is a kind of albumen on Staphylococcus aureus cell membrane
Matter, Fc areas that can be specifically with mammalian antibody (mainly IgG) are combined, and Protein G is Streptococcal cells wall-held protein, energy
It is enough to be combined with the constant region of IgG antibody, albumin, alpha2-macroglobulin.Therefore, purified using the affine filler of albumin A, Protein G
Obtained antibody belongs to mixed antibody, this antibody it is specific poor.
Typical method using albumin A, protein g affinity chromatography antibody purification is as follows：(1) 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 obtain activation and fill out
Material；(2) albumin A of purifying or Protein G are added in Activation filling, by being covalently bound to filler, prepares affine fill out
Material；(3) make filler of the thick solution of antiserum Jing Guo fixed protein A goods Protein G, IgG is adsorbed onto on filler；(4) with neutrality
Wash buffer filler, removes other impurities；(4) using acidic buffer (pH2.5~pH3.0) elution absorption on filler
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, affinity can be obtained applied to antibody purification
The high antibody of good, high specificity, purity.(2015103209206) are invented by the denatured antigen albumen (inclusion body of such as prokaryotic expression
Albumen) solid phase carrier is directly coupled at antibody purification, operating procedure is simplified, that is, prepares soluble antigen protein process, institute
Obtaining antibody has high purity and specificity, is suitable for laboratory and prepares and applied in industrial production.The swine fevers such as document envelope beautiful jade
The Sepharose-4B couplings of viral antigen and Epichlorohydrin activation, being prepared into immune affinity chromatographic column is used for the antiviral antibody
Purifying, obtain the antiviral antibody that purity is high, activity is strong.But, due to using holoantigen, obtained antibody identification is purified
Fall within mixed antibody, it is impossible to for pathogen strain, the precise Identification of isolate.
In recent years, people going deep into research work, it is found that the cymbidium mosaic virus for infecting orchid has substantial amounts of strain
System, the variation of isolate, these variations cause the pathogenic of different strains to there is bigger difference, wherein strong pathogenic strain can result in
More serious illness, and low virus influence orchid degree is relatively low.Furthermore, with further going deep into for research, it has also been found that weak
The metainfective orchid of diseased plant can slow down the infection harm of strong diseased plant, there is a certain degree of cross-protection；On the other hand,
People also have found many unknown viral species from orchid diseased plant, wherein have part and cymbidium mosaic virus closely similar, one
Directly it is taken as the virus to be prevented and treated, causes preventing and controlling to be got half the result with twice the effort.It can be seen that, precise Identification virus and virus strain are for anti-
Control that work is most important, the purification technique of existing antibody can not carry out finer purifying to antibody,.
Given this it can be used in a series of polypeptides of purification specificity antibody from antiserum the invention provides a kind of and match somebody with somebody
Base affinity media, the need for meeting antibody purification.
The content of the invention
It is an object of the invention to provide a kind of affine filler of cymbidium mosaic virus antibody and preparation method thereof, it can meet
The need for antibody purification.
The present invention uses following technical scheme：
A kind of preparation method of the affine filler of cymbidium mosaic virus antibody, is repaiied using amino silane to filler surface
After decorations, fabricated in situ linking arm, the aglucon further modified is the polypeptide fragment of cymbidium mosaic virus coat protein different zones,
Enable the specific antibody of filler absorbing shell albumen different zones.
The polypeptide fragment is located at the fragment of the special conserved region of virus for selection, further analyzes these fragment amino acids residual
Hydrophily, surface accessibility and the antigenicity of base.
Polypeptide fragment is to include CP albumen n ends to C-terminal, and most short is 6 amino acid lengths, up to 30 amino acid lengths
A series of polypeptide fragments.
Described polypeptide aglucon corresponds to the polypeptide fragment of cymbidium mosaic virus coat protein different zones, and length is 6-30
Individual amino acid residue.
Using 0.1%-20% amino silane solution, it is added in filler in the NH for being stored at room temperature 30min-12h progress fillers2
Modification.
Using Fmoc methods or Boc methods on surface with amido modified filler or other with NH2Carried out on group filler
Fabricated in situ polypeptide, obtains that " G (n) S " polypeptides are connected, and wherein n represents G quantity, respectively 3-20, and its end carries NH2Base
Group.
Polypeptide fragment is coupled into filler by coupling method can equally be prepared into the affine filler of identical.
Coupling method is any one in glutaraldehyde method, active ester method, carbodlimide method, succinic anhydride method and diazotising method.
After antiserum is diluted with 0.05M-0.2M PBS, add in affine filler and stand 1-3 hours, it is then molten with PBS
Liquid washing removes nonspecific antibody and other impurities；PH3.0-4.0 glycine-HCI buffer solution, citrate buffer solution is used again
Or other elution solution carry out the elution of antibody, by a series of antibody-solutions NaOH, NaCO for eluting3Deng alkaline solution
PH is adjusted to neutrality, the antibody purified after desalination of dialysing.
Filler is in cellular glass, glass fibre, silica gel, resin, Ago-Gel, magnetic particle material, cellulose
Any one.
A kind of affine filler of cymbidium mosaic virus antibody its prepared by above-mentioned preparation method.
It is an advantage of the invention that：The affine filler of cymbidium mosaic virus antibody of the present invention can be used in purifying and be directed to shell egg
The detection antibody of white different zones, can be used for Viral diagnosis work, can be used for virus strain, the difference identification work of isolate
Make.
Embodiment
The purpose of the present invention is to utilize cymbidium mosaic virus coat protein amino acid sequence information, synthesizes a series of polypeptide pieces
Duan Zuowei ligand cous prepare a series of affine filler to filler, and the described affine filler of cymbidium mosaic virus antibody can be from
Purifying obtains the antibody for different fragments in cymbidium mosaic virus antiserum.This method, which can be purified, obtains specific for virus
The detection antibody of amino acid residue, its specificity is far above the antibody that albumin A, protein g affinity chromatography are purified, and operation is simple
Just it is, simple to equipment requirement.
In order to realize the purpose, the technical scheme is that：
According to cymbidium mosaic virus coat protein amino acid sequence, preferably with certain hydrophily, surface accessibility or anti-
Immunogenic sequence, design length is a series of polypeptide fragments of 6-30 amino acid residue；Using amino silane to including porous
Glass, glass fibre, silica gel, resin, Ago-Gel, magnetic particle material, cellulose (such as cotton, fiber chromatographic paper) carry out ammonia
Baseization modification obtains amido modified filler；Connection is further synthesized in above-mentioned amido modified filler using conventional Fmoc or Boc methods
Arm " GnS " (wherein n represents G quantity, respectively 3-20), its end carries NH2 groups；With above-mentioned surface modification filler or
Other common fillers with NH2 groups are base material, and the fabricated in situ for carrying out polypeptide fragment using Fmoc methods or Boc methods obtains many
Peptide aglucon is affine filler；The present invention also includes obtaining after polypeptide fragment using conventional chemical synthesis, passes through glutaraldehyde method, activation
Polypeptide fragment is coupled to filler system by ester process, carbodlimide method, succinic anhydride method and diazotising method or other common coupling methods
It is standby to obtain affine filler.According to polypeptide and the relatively low characteristic of affinity of antibody, in antibody purification procedures, preferably pH3.5-4.5
Elution buffer, so as to reduce the destruction to antibody activity.
Experimental procedure by the invention is：
1) design of polypeptide fragment
First according to CyMV CP conserved amino acid sequences, selection is located at the fragment of the special conserved region of virus；Further analysis
Hydrophily, surface accessibility and the antigenicity of these fragment amino acid residues；CP albumen n ends are preferably included to C-terminal, most short is 6
A series of polypeptide fragments of individual amino acid length, up to 30 amino acid lengths.
2) filler is amido modified
Using 0.1%-20% amino silane solution, it is added in filler in the NH2 for being stored at room temperature 30min-12h progress fillers
Modification.Using conventional Fmoc or Boc methods in the enterprising one-step synthesis linking arm " GnS " of filler that NH2 is modified.
3) fabricated in situ of polypeptide fragment
Using Fmoc methods or Boc methods on above-mentioned surface with amido modified filler or other with NH2 group fillers
Fabricated in situ polypeptide is carried out, described polypeptide is above-mentioned preferred polypeptide.
4) fixation of polypeptide fragment aglucon
After the synthesis that above-mentioned preferred polypeptide fragment is carried out using chemical synthesis, using glutaraldehyde method, active ester method, carbon
Polypeptide fragment is coupled to filler by diimine method, succinic anhydride method and diazotising method or other common coupling methods can equally make
It is standby to arrive the affine filler of identical.
5) purifying of antibody
After antiserum is diluted with 0.05M-0.2M PBS, it is added in a series of affine fillers prepared by the present invention and stands
1-3 hours, removal nonspecific antibody and other impurities are then washed with PBS solution；PH3.0-4.0 glycine-HCI is used again
Buffer solution, citrate buffer solution or other elution solution carry out the elution of antibody.A series of antibody-solutions eluted are used
The alkaline solutions such as NaOH, NaCO3 adjust pH to neutrality, and a series of antibody of purifying is obtained after desalination of dialysing.
Main advantages of the present invention are can to obtain a series of antibody for the different protein fragments of virus by purifying, and
And the activity of antibody can be ensure that using relatively mild elution requirement.
Embodiment one
Exemplified by this embodiment is CyMV CP " TDRERAAHS " polypeptide fragment, description affine is filled out for the antibody of the fragment
The preparation method of material；And the specificity experiments of antibody purification and albumin A affinity purification antibody are further compared, illustrate the present invention
The advantage of affine filler.
Method used in following embodiments is conventional method unless otherwise specified.
Material, reagent used in following embodiments etc., unless otherwise specified, are commercially obtained.
1 material
1.1 major experimental equipment and consumptive material
Glass fibre is bought for market；Using 3 intramuscular injection and 1 intravenous immune program, with sword-leaved cymbidium mosaic disease
Poison is immunogen immune rabbit, determines potency up to 1:Rabbit anti-serum is gathered after 25000.
2 methods
The surface NH of 2.1 glass fibres2Modification
10g glass fibres are taken, with the concentrated sulfuric acid and 30% hydrogen peroxide v/v3:1 immersion 5min, is cleaned to neutrality, 120 with pure water
DEG C heated-air drying.After glass fibre is cooled to room temperature, adds 5% amino silane ethanol solution and soak 4 hours, be filtered dry
Afterwards, with 10 times of volume pure water rinsings, 80 DEG C of heated-air dryings obtain amido modified fiber glass packing.
The fabricated in situ of 2.2 polypeptide fragments
Use Fmoc methods to synthesize the serine of 1 equivalent based on the NH2 groups on filler surface, first, further close
Into 3 continuous Formation of glycine " GGGS " polypeptide linking arms, the corresponding polypeptide fragments of CyMV CP 171-179aa are finally synthesizing
" TDRERAAHS ", forms the affinity chromatography medium of " TDRERAAHSGGGS- glass fibres ".Amino and carboxyl condensation of the present invention is anti-
It should be condensing agent using MHBTU, DIEPC, be carried out using conventional solid synthetic method.
2.3 antibody purification
Affine filler is added in 50mL affinity columns, it is standby after being balanced with 100mL 0.1M PBS；It is dilute using 20mL PBS
It is added to after releasing 1mL antiserums in affinity column, 37 DEG C of incubations, 1 hour absorption specificity antibody；Eluted and removed using 100mL PBS
Non-specific antibody and impurity；Reuse after the elution of 0.005M phosphoric acid solutions, be neutralized to neutrality with 2 times of volume 0.005NaOH, adopt
Antibody purification 1mL is finally given with PEG8000 dehydrations and pellicle dialysis.
The regeneration of 2.4 affinity columns
Affine filler is rinsed with 10 times of volume 0.01M phosphoric acid, again with pure water rinsing to neutrality, contain 20% second with 5 times of volumes
The PBS balance affinity columns of alcohol, are stored in 4 DEG C.
2.5 antibody purification concentration mensurations
Using BSA as standard, standard curve is drawn using Coomassie brilliant G-250 method, antibody purification concentration mensuration is carried out.
2.6 antibody titers are determined
With 1ng/mL virion coated elisa plate, and BSA is coated with for negative control, using antiserum before purification and pure
Antibody determines antibody titer respectively after change.
2.7 antibody specificities are tested
Infection CyMV Phalaenopsis leaves, healthy iris leaf are detected respectively using antiserum before purification and purified antibodies
The samples such as piece, potato virus X positive control and healthy potato leaf, compare purification experiment to the specific influence of antibody.
3 results
3.1 antibody purification concentration
Use Coomassie brilliant G-250 method to determine and show the concentration of antibody purification for 10ng/mL, about antiserum concentration
1/1000, show that purification experiment eliminates substantial amounts of non-specific protein.
3.2 antibody titers are determined
The potency of antiserum and antibody purification before purification is determined by indirect ELISA, as a result shows antiserum effect before purification
Valency is 1:25000；Antibody titer 1 after purification:5000.
3.3 antibody specificities are tested
Before purification there is obvious compatible reaction, the obvious (A of chromogenic reaction in antiserum with CyMV positives405=0.487
± 0.024), but for occurring in that certain cross reaction (A405=0.0.288 when detecting potato virus X positive
± 0.004), and the higher (A of detection healthy sample colour developing colour developing background405=0.147 ± 0.034), it have impact on testing result
Evaluation；With CyMV positives obvious compatible reaction occurs for antibody after purification, although chromogenic reaction has lowered (A405=
0.325 ± 0.002), but do not occur cross reaction (A with healthy Phalaenopsis leaves405=0.024 ± 0.010), also not with horse
Cross reaction (A occurs for bell potato X virus positive controls405=0.031 ± 0.017).
Embodiment two
This embodiment be CyMV CP "186RQRIQNGNLITN200" exemplified by polypeptide fragment, description is for the anti-of the fragment
The preparation method of body is affine filler；And the specificity experiments of antibody purification and albumin A affinity purification antibody are further compared, say
The advantage of bright affine filler of the invention.
Method used in following embodiments is conventional method unless otherwise specified.
Material, reagent used in following embodiments etc., unless otherwise specified, are commercially obtained.
1 material
1.1 major experimental equipment and consumptive material
100 mesh silica gel are bought for market；Using 3 intramuscular injection and 1 intravenous immune program, with sword-leaved cymbidium mosaic disease
Poison is immunogen immune rabbit, determines potency up to 1:Rabbit anti-serum is gathered after 25000.
2 methods
The surface NH2 modifications of 2.1 glass fibres
The mesh silica gel of 10g 100 is taken, with the concentrated sulfuric acid and 30% hydrogen peroxide v/v3:1 immersion 5min, is cleaned to neutrality with pure water,
120 DEG C of heated-air dryings.After silica gel is cooled to room temperature, adds 5% amino silane ethanol solution and soak 4 hours, after being filtered dry,
With 10 times of volume pure water rinsings, 80 DEG C of heated-air dryings obtain amido modified silica filler.
The fabricated in situ of 2.2 linking arms
Use Fmoc methods to synthesize the serine of 1 equivalent based on the NH2 groups on filler surface, first, further close
Into 3 continuous Formation of glycine " GGGS " polypeptide linking arms, its end carries NH2 groups.
2.3 obtain 10mg polypeptide fragment " RQRIQNGNLITN " using chemical synthesis, are coupled to and filled out by glutaraldehyde method
Expect linking arm NH2, obtain the affinity chromatography medium of " RQRIQNGNLITNGGGS- silica gel ".
2.4 antibody purification
Affine filler is added in 50mL affinity columns, it is standby after being balanced with 100mL 0.1M PBS；It is dilute using 20mL PBS
It is added to after releasing 1mL antiserums in affinity column, 37 DEG C of incubations, 1 hour absorption specificity antibody；Eluted and removed using 100mL PBS
Non-specific antibody and impurity；Reuse after the elution of 0.005M phosphoric acid solutions, be neutralized to neutrality with 2 times of volume 0.005NaOH, adopt
Antibody purification 1mL is finally given with PEG8000 dehydrations and pellicle dialysis.
The regeneration of 2.4 affinity columns
Affine filler is rinsed with 10 times of volume 0.01M phosphoric acid, again with pure water rinsing to neutrality, contain 20% second with 5 times of volumes
The PBS balance affinity columns of alcohol, are stored in 4 DEG C.
2.5 antibody purification concentration mensurations
Using BSA as standard, standard curve is drawn using Coomassie brilliant G-250 method, antibody purification concentration mensuration is carried out.
2.6 antibody titers are determined
With 1ng/mL virion coated elisa plate, and BSA is coated with for negative control, using antiserum before purification and pure
Antibody determines antibody titer respectively after change.
2.7 antibody specificities are tested
Detect infection CyMV iris leaf respectively using Protein A purification antibody and affine filler antibody purification of the invention
The samples such as piece, healthy Phalaenopsis leaves, potato virus X positive control and healthy potato leaf, compare purification experiment confrontation
The specific influence of body.
3 results
3.1 antibody purification concentration
Use Coomassie brilliant G-250 method to determine and show the concentration of antibody purification for 10ng/mL, about antiserum concentration
1/1000, show that purification experiment eliminates substantial amounts of non-specific protein.
3.2 antibody titers are determined
The potency of antiserum and antibody purification before purification is determined by indirect ELISA, as a result shows antiserum effect before purification
Valency is 1:25000；Antibody titer 1 after purification:4000.
3.3 antibody specificities are tested
There is obvious compatible reaction, the obvious (A of chromogenic reaction with CyMV positives in Protein A purification antibody405=0.544
± 0.015), but for occurring in that certain cross reaction (A when detecting potato virus X positive405=0.287 ±
, and detection healthy sample colour developing, colour developing background higher (A 0.047)405=0.172 ± 0.044), it have impact on testing result
Evaluation；With CyMV positives obvious compatible reaction occurs for antibody after purification, although chromogenic reaction has lowered (A405=
0.417 ± 0.011), but do not occur cross reaction (A with healthy Phalaenopsis leaves405=0.031 ± 0.005), also not with horse
Cross reaction (A occurs for bell potato X virus positive controls405=0.011 ± 0.001).
Embodiment three
This embodiment be CyMV CP "180IGKYGALARQRIQNGNLITNIAEVTKGHLG209" exemplified by polypeptide fragment,
Preparation method of the description for the affine filler of antibody of the fragment；And further compare antibody purification and albumin A affinity purification
The specificity experiments of antibody, illustrate the advantage of affine filler of the invention.
Method used in following embodiments is conventional method unless otherwise specified.
Material, reagent used in following embodiments etc., unless otherwise specified, are commercially obtained.
1 material
1.1 major experimental equipment and consumptive material
CNBr activated sepharoses；Using 3 intramuscular injection and 1 intravenous immune program, with sword-leaved cymbidium mosaic disease
Poison is immunogen immune rabbit, determines potency up to 1:Rabbit anti-serum is gathered after 25000.
2 methods
2.1 Peptide systhesis
10mg polypeptide fragments " IGKYGALARQRIQNGNLITNIAEVTKGHLG " are obtained by the synthesis of Fmoc methods, are dissolved in
In 10ml pH9.0 0.25M sodium carbonate buffers.10mL fillers are added, are vibrated on shaking table, 25 DEG C are coupled 6 hours.Then
Plus 50mg glycine, then 6 remaining groups of hours closing are reacted, obtain affine filler.
2.3 antibody purification
Affine filler is added in 50mL affinity columns, it is standby after being balanced with 100mL 0.1M PBS；It is dilute using 20mL PBS
It is added to after releasing 1mL antiserums in affinity column, 37 DEG C of incubations, 1 hour absorption specificity antibody；Eluted and removed using 100mL PBS
Non-specific antibody and impurity；Reuse after the elution of 0.005M phosphoric acid solutions, be neutralized to neutrality with 2 times of volume 0.005NaOH, adopt
Antibody purification 1mL is finally given with PEG8000 dehydrations and pellicle dialysis.
The regeneration of 2.4 affinity columns
Affine filler is rinsed with 10 times of volume 0.01M phosphoric acid, again with pure water rinsing to neutrality, contain 20% second with 5 times of volumes
The PBS balance affinity columns of alcohol, are stored in 4 DEG C.
2.5 antibody purification concentration mensurations
Using BSA as standard, standard curve is drawn using Coomassie brilliant G-250 method, antibody purification concentration mensuration is carried out.
2.6 antibody titers are determined
With 1ng/mL virion coated elisa plate, and BSA is coated with for negative control, using antiserum before purification and pure
Antibody determines antibody titer respectively after change.
2.7 antibody specificities are tested
Infection CyMV Phalaenopsis leaves, healthy iris leaf are detected respectively using antiserum before purification and purified antibodies
The samples such as piece, potato virus X positive control and healthy potato leaf, compare purification experiment to the specific influence of antibody.
3 results
3.1 antibody purification concentration
Use Coomassie brilliant G-250 method to determine and show the concentration of antibody purification for 10ng/mL, about antiserum concentration
1/1000, show that purification experiment eliminates substantial amounts of non-specific protein.
3.2 antibody titers are determined
The potency of antiserum and antibody purification before purification is determined by indirect ELISA, as a result shows antiserum effect before purification
Valency is 1:25000；Antibody titer 1 after purification:5000.
3.3 antibody specificities are tested
Before purification there is obvious compatible reaction, the obvious (A of chromogenic reaction in antiserum with CyMV positives405=0.456
± 0.004), but for occurring in that certain cross reaction (A when detecting potato virus X positive405=0.245 ±
, and detection healthy sample colour developing colour developing background higher (A 0.045)405=0.178 ± 0.054), it have impact on commenting for testing result
It is fixed；With CyMV positives obvious compatible reaction occurs for antibody after purification, although chromogenic reaction has lowered (A405=0.387
± 0.021), but do not occur cross reaction (A with healthy Phalaenopsis leaves405=0.043 ± 0.010), also not with potato X
Cross reaction (A occurs for virus positive control405=0.015 ± 0.001).
Although it is relatively low to purify obtained antibody yield as can be seen here, potency is caused to decline, obtained antibody specificity
Significantly risen, be more beneficial for improving the accuracy of detection.
Traditional albumin A, Protein G affine technolog, using albumin A, Protein G as aglucon, are coupled to filler and prepare and build parent
And filler, albumin A, Protein G can Specific adsorption antibody (IgG), can be purified from antiviral serum obtain all IgG resist
Body, is a kind of mixed antibody.It is a kind of mixture to purify obtained IgG, contains special viral antibody and other a large amount of albumen
Antibody.This antibody can occur nonspecific reaction to foreign protein, similar virus, cause detection accurate in application process
Property is poor.
" the immune-affinity chromatography fast purifying antibody against swine fever virus " of prior art, using pathogen holoprotein as with
Base, is coupled to filler and prepares affine filler, and it utilizes Ag-Ab compatible reaction, can purify and obtain from antiviral serum
The IgG antibody of viral all epitopes is detected, non-specific antibody can be effectively removed.This antibody will not be miscellaneous with other
Nonspecific reaction occurs for albumen, and purifying, which obtains IgG antibody, to be reacted with viral all epitopes.Therefore, it is this
Antibody is easily influenceed, so as to cause false positive to occur in application process by other similar or close viral species.
The antigen epitope polypeptide that the present invention uses cymbidium mosaic virus (CyMV) particle surface is coupled to filler as aglucon
Affine filler is prepared, using antigen epitope polypeptide-antibody compatible reaction, can be purified from antiviral serum and obtain specific antigen
The IgG antibody of epitope, is a kind of monospecific antibody, and the specific fragment of virus can be detected by purifying obtained antibody, and this antibody is not
Reacted to other similar, close viral and other foreign proteins, accuracy is higher.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
Any modifications, equivalent substitutions and improvements made within refreshing and principle etc., should be included in the scope of the protection.
Claims (10)
1. a kind of preparation method of the affine filler of cymbidium mosaic virus antibody, it is characterised in that：Comprise the following steps：
Filler surface is modified using amino silane, after fabricated in situ linking arm, the aglucon further modified is sword-leaved cymbidium
The polypeptide fragment of mosaic virus coat protein different zones, enables the specific antibody of filler absorbing shell albumen different zones.
2. preparation method according to claim 1, it is characterised in that：The polypeptide fragment is located at virus for selection specifically to be protected
The fragment of defending zone, further analyzes the hydrophily, surface accessibility and antigenicity of these fragment amino acid residues.
3. preparation method according to claim 2, it is characterised in that：Polypeptide fragment be cymbidium mosaic virus coat protein not
With the polypeptide fragment in region, length is 6-30 amino acid residue.
4. preparation method according to claim 1, it is characterised in that：Using 0.1%-20% amino silane solution, it is added to
In the NH for being stored at room temperature 30min-12h progress fillers in filler2Modification.
5. preparation method according to claim 1, it is characterised in that：Amino is carried on surface using Fmoc methods or Boc methods
The filler of modification is other with NH2Fabricated in situ polypeptide is carried out on group filler, " connection of G (n) S " polypeptides, wherein n generations is obtained
Table G quantity, respectively 3-20, its end carry NH2Group.
6. preparation method according to claim 1, it is characterised in that：Polypeptide fragment is coupled to by filler by coupling method same
Sample can be prepared into the affine filler of identical.
7. preparation method according to claim 6, it is characterised in that：Coupling method is glutaraldehyde method, active ester method, the Asia of carbon two
Any one in amine method, succinic anhydride method and diazotising method.
8. preparation method according to claim 1, it is characterised in that：After antiserum is diluted with 0.05M-0.2M PBS,
Add in affine filler and stand 1-3 hours, removal nonspecific antibody and other impurities are then washed with PBS solution；Use again
PH3.0-4.0 glycine-HCI buffer solution, citrate buffer solution or other elution solution carry out the elution of antibody, will elute
A series of antibody-solutions NaOH, the NaCO got off3PH is adjusted to neutrality Deng alkaline solution, and what is purified after desalination of dialysing is anti-
Body.
9. preparation method according to claim 1, it is characterised in that：Filler is cellular glass, glass fibre, silica gel, tree
Any one in fat, Ago-Gel, magnetic particle material, cellulose.
10. a kind of affine filler of cymbidium mosaic virus antibody, it passes through any one described preparation method in claim 1 to 9
Prepare.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710526035.2A CN107091928A (en) | 2017-06-30 | 2017-06-30 | A kind of affine filler of cymbidium mosaic virus antibody and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710526035.2A CN107091928A (en) | 2017-06-30 | 2017-06-30 | A kind of affine filler of cymbidium mosaic virus antibody and preparation method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107091928A true CN107091928A (en) | 2017-08-25 |
Family
ID=59641157
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710526035.2A Pending CN107091928A (en) | 2017-06-30 | 2017-06-30 | A kind of affine filler of cymbidium mosaic virus antibody and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107091928A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109870575A (en) * | 2017-12-04 | 2019-06-11 | 厦门华侨亚热带植物引种园 | A method of improving papaya ringspot virus antibody specificity |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5153166A (en) * | 1988-08-18 | 1992-10-06 | Trustees Of At Biochem | Chromatographic stationary supports |
| US5451683A (en) * | 1989-11-13 | 1995-09-19 | Affymax Technologies N.V. | Spatially-addressable immobilization of anti-ligands on surfaces |
| CN101748136A (en) * | 2010-01-15 | 2010-06-23 | 浙江大学 | Expression of cymbidium mosaic virus coat protein gene and preparation method for antibody of cymbidium mosaic virus coat protein gene |
| CN102757493A (en) * | 2012-07-11 | 2012-10-31 | 北京健翔和牧生物科技有限公司 | Method for preparing chicken pox virus antibody |
| CN103111093A (en) * | 2012-11-23 | 2013-05-22 | 南宁市蓝光生物技术有限公司 | Preparation and application of flag peptide tagged recombinant protein immunoaffinity purification and enrichment column |
| CN103788210A (en) * | 2012-10-29 | 2014-05-14 | 汕头大学医学院 | Specific antibody of phosphorylated aromatase |
| CN105498706A (en) * | 2015-12-09 | 2016-04-20 | 广东研捷医药科技有限公司 | Preparation method for protein A immunoaffinity column |
-
2017
- 2017-06-30 CN CN201710526035.2A patent/CN107091928A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5153166A (en) * | 1988-08-18 | 1992-10-06 | Trustees Of At Biochem | Chromatographic stationary supports |
| US5451683A (en) * | 1989-11-13 | 1995-09-19 | Affymax Technologies N.V. | Spatially-addressable immobilization of anti-ligands on surfaces |
| CN101748136A (en) * | 2010-01-15 | 2010-06-23 | 浙江大学 | Expression of cymbidium mosaic virus coat protein gene and preparation method for antibody of cymbidium mosaic virus coat protein gene |
| CN102757493A (en) * | 2012-07-11 | 2012-10-31 | 北京健翔和牧生物科技有限公司 | Method for preparing chicken pox virus antibody |
| CN103788210A (en) * | 2012-10-29 | 2014-05-14 | 汕头大学医学院 | Specific antibody of phosphorylated aromatase |
| CN103111093A (en) * | 2012-11-23 | 2013-05-22 | 南宁市蓝光生物技术有限公司 | Preparation and application of flag peptide tagged recombinant protein immunoaffinity purification and enrichment column |
| CN105498706A (en) * | 2015-12-09 | 2016-04-20 | 广东研捷医药科技有限公司 | Preparation method for protein A immunoaffinity column |
Non-Patent Citations (6)
| Title |
|---|
| ANDREA MURRAY ET AL: "Purification of monoclonal antibodies by epitope and mimotope affinity chromatography", 《JOURNAL OF CHROMATOGRAPHY A》 * |
| MARTINEZ-CERON ET AL: "Recombinant protein purification using complementary peptides as affinity tags", 《NEW BIOTECHNOLOGY》 * |
| 明艳林 等: "建兰花叶病毒研究进展", 《福建农业学报》 * |
| 熊丽 等: "《生物化学实验教程》", 31 August 2011 * |
| 王捷: "《动物细胞培养技术与应用》", 31 May 2004 * |
| 田亚平 等: "《生化分离技术》", 30 September 2006 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109870575A (en) * | 2017-12-04 | 2019-06-11 | 厦门华侨亚热带植物引种园 | A method of improving papaya ringspot virus antibody specificity |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| AU2002254683C1 (en) | Binding molecules for Fc-region polypeptides | |
| KR0181343B1 (en) | Synthetic antigens for the detection of antibodies to hepatitis c virus | |
| AU2002254683A1 (en) | Binding molecules for Fc-region polypeptides | |
| JP2008101023A (en) | IgG SEPARATION MEDIUM AND NOVEL PROTEIN A VARIANT | |
| US20220112236A1 (en) | Immunoglobulin binding proteins for affinity purification | |
| JPH04505857A (en) | Monoclonal antibody against C-reactive protein | |
| CN106146627B (en) | Fc Specific binding proteins, IgG affinity chromatography medium and the preparation method and application thereof | |
| CN100486993C (en) | Evolved immunoglobulin binding molecule, and its preparation method and uses | |
| EP1328811A2 (en) | Hcv mosaic antigen composition | |
| EP2614371B1 (en) | Method for labeling of compounds | |
| CN107091928A (en) | A kind of affine filler of cymbidium mosaic virus antibody and preparation method thereof | |
| CA1341014C (en) | Hcg peptides for use in antibody purification procedures | |
| CN103113455B (en) | Preparation and application of hemagglutinin peptide mark recombinant protein immunoaffinity purification enriching column | |
| CN109870575A (en) | A method of improving papaya ringspot virus antibody specificity | |
| CN103254294B (en) | CD34-SG17 polypeptide fragment as well as preparation method and application thereof | |
| CN105859838B (en) | A kind of Escherichia coli O 157: H7 protein I vy polypeptide, anti-Ivy polyclonal antibody and its application | |
| JPH04505623A (en) | Cysteine thiol protected peptides for use in immunoassays | |
| US5436127A (en) | Epitope-related peptides of human parvovirus | |
| US5254672A (en) | Synthetic peptides which contain sequences from factor VIIa, and the use thereof | |
| CN103111093B (en) | The preparation of flag peptide tag recombinant protein immunoaffinity purification enriching column and application thereof | |
| ITMI952582A1 (en) | IMPROVED ANTIGENIC PEPTIDES | |
| CN108707186B (en) | Human sperm specific antigen epitope peptide, polymer and application thereof | |
| JP2023533529A (en) | Immunoglobulin binding proteins for affinity purification | |
| WO2003060086A2 (en) | Napi type iib polypeptides and methods for making and using same | |
| RU2304146C2 (en) | Method for stabilizing loop-shaped structure of synthetic peptides using n- and c-terminal modification |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170825 |
|
| RJ01 | Rejection of invention patent application after publication |