CN109870575A

CN109870575A - A method of improving papaya ringspot virus antibody specificity

Info

Publication number: CN109870575A
Application number: CN201711257102.1A
Authority: CN
Inventors: 张文珠; 郑国华; 林德钦
Original assignee: XIAMEN OVERSEAS CHINESE SUBTROPICAL PLANT INTRODUCTION GARDEN
Current assignee: XIAMEN OVERSEAS CHINESE SUBTROPICAL PLANT INTRODUCTION GARDEN
Priority date: 2017-12-04
Filing date: 2017-12-04
Publication date: 2019-06-11

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

A method of improving papaya ringspot virus antibody specificity

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.

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 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

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.