CN102827273A - Preparation of vaccinia virus (VACA) resistant egg yolk antibody (IgY) - Google Patents
Preparation of vaccinia virus (VACA) resistant egg yolk antibody (IgY) Download PDFInfo
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Abstract
The invention discloses preparation of a vaccinia virus (VACA) resistant egg yolk antibody (IgY) and a method for enriching the VACA by utilizing an antibody-magnetic bead coupling substance, belonging to the field of biotechnology, which can be applied to detecting the VACA and treatment of infection. The VACA resistant egg yolk antibody is a diagnosis agent prepared by getting the inactivated VACA as an antigen immunization layer, using a polyethyleneglycol (PEG)-6000 precipitation method to obtain the antibody from egg yolk so as to prepare a medicine for preventing the infection of the VACA, and enriching the VACA through coupled magnetic beads. An experiment shows that the specific VACA resistant egg yolk antibody has the capacity of combining specificity with corresponding VACA antigen to prevent the infection of the VACA; and the egg yolk antibody under the coupled magnetic beads is adopted to enrich the VACA, and can be served as a diagnosis tool for the VACA.
Description
Technical field
(preparation method of particularly anti-vaccinia virus IgY antibody and this antibody thereof are used for the purposes of vaccinia virus immune diagnostic reagent for egg yolk antibody, preparation IgY) and application to the invention belongs to a kind of special yolk antibody in the biological technical field.
Background technology
(Vaccinia Virus VACA) belongs to orthopoxvirus (Orthopoxvirus) member to vaccinia virus, can be used as the vaccine prevention smallpox, can be used as the model of experiment with poxvirus simultaneously.But propagate in Domestic Animal and the laboratory animal under the vaccinia virus state of nature, cause that the nipple of ox infects and immunity.Vaccinia virus also can cause people's infection, especially population at risk's (like pregnant woman, immunodeficient person, dermatosis patient) is infected to cause severe complications.The people can obtain the immunizing power to variola virus after inoculating the vaccinia virus vaccine, but along with the extinction of sky blossom disease in the whole world, poxvirus vaccine is abandoned using in a lot of countries and crowd.Owing to multiple reasons such as the close contacts of people and animals; At present in people and relevant animal species; The infection of vaccinia subgroup virus and morbidity are obvious ascendant trend, and the antibody and the detection reagent thereof that obtain relevant poxvirus are significant for the diagnosis and the neutrality treatment of this pathogenic agent in physianthropy and animal doctor.
IgY antibody is through to bird (chicken) immunization specific antigen, thereby produces IgY in vivo, be delivered in the yolk by parent through blood, and enrichment in yolk, and then through separating yolk, the specific polyclonal antibody of acquisition.With respect to from mammalian blood serum, obtaining antibody, avoided conventional animal blood sampling, alleviated the misery of animal; Obtain antibody through yolk simultaneously, output is big, and preparation is simple; Can purify in the every piece of egg antibody of 50～100mg, and hen is easy to raise, and expense is low; Be convenient to produce on a large scale specific antibody, economic advantages are obvious.IgG is similar with Mammals, and the Fab fragment of IgY comprises antigen binding site, and there are functions such as complement activation function, opsonification and sensitization anaphylaxis effect in the site that the Fc fragment comprises.But its structure is with some difference of Mammals, and with respect to IgG, the amino-acid residue quantity of IgY heavy chain is more than IgG, and CH (CH district) quantity is more than IgG, and light chain is less than IgG.IgY physicochemical property and resistance to enzymolysis characteristic etc. are different from IgG, make IgY be more suitable for being used to treat preparation with respect to IgG.IgY iso-electric point (PI) is between 5.7-7.6, and some IgY is stable in pH4.0～11.0, and is more more responsive to sour sex change than rabbit igg, when pH value when 4 drop to 3, active rapid forfeiture of IgY, and IgG receives minor impact to this; Performance was more stable when IgY stored at low temperatures; Can keep 6 months activity under the room temperature; 4 ℃ store 6～7 years active only 5% (Jaradat ZW that descend; Marquardt R is on the stability of chicken IgY in different sugars R.2000.Studies, complex carbohydrates and food materials.Food andAgricultural Immunology, 12 (4): 263-272); The stability of IgY antipepsin digestion is poor than ox IgG; But then strong (the Shimizu M of antitrypsin and Chymotrypsin ability; Nagashima H; Sano K, et al.1992.Molecular stability of chicken and rabbit immunoglobulin G. Biosci Biotechnol Biochem, 56 (2): 270-274).Because the phylogenetics distance causes Mammals and the specific difference of chicken antibody; With respect to Mammals IgG; Yolk antibody be applied to detect also have many-sided advantage as: the yolk antibody and the rheumatism factor, human anti-mouse antibody (HAMA) do not have cross reaction; Active and the heterogeneous lectin of no complement system, and debond albumin A and Protein G can be avoided cross reaction.In addition, bird often can cause than Mammals antibody intensive antibody response more to the strong protein of conservative property, becomes the important selection factor of relevant antibody research and development.
Summary of the invention
First purpose of the present invention provides a kind of preparation method of anti-VACA yolk antibody.
Second purpose of the present invention provides the external neutralizing effect of the anti-VACA-IgY of preparation to the VACA infection.
The 3rd purpose of the present invention provides the method with the anti-VACA-IgY antibody coupling immunomagnetic beads of preparation, so that the detection thing that contains VACA is carried out enrichment, is used to detect VACA.
In order to realize above-mentioned task, the present invention takes following technical scheme:
The preparation method of 1 anti-VACA yolk antibody
(1) antigen prepd
Many inoculations of a:VACA instar chicken embryo tire inoblast (CEF), after hatching, ultracentrifugation is suspended from the PBS damping fluid;
B: Superlysoform deactivation VACA, store-70 ℃ subsequent use.
(2) immunization method
Initial immunity is with Freund emulsive antigen intramuscular injection immunity white Leghorn in 16 age in week, immunizing dose 400 μ g; Just exempt to exempt from back 36 days two Shi Yongfeishi Freund, immunizing dose 400 μ g; Three exempt from after 72 days, immunizing dose 2000 μ g; Exempted from immunizing dose 1000 μ g in 148 days four; Exempted from immunizing dose 2000 μ g in 319 days five; Exempted from immunizing dose 2000 μ g in 389 days six.Collect egg every day, make marks, 4 ℃ of preservations.
(3) purifying of IgY
The separation of IgY and purifying adopt polyethylene glycol precipitation, and method is following:
A: separate yolk and egg white, collect yolk, use PBS (pH 7.4) to dilute in yolk according to 1: 2;
B: add the PEG-6000 of 3.5% (W/V), fully stir room temperature effect 10min on the shaking table;
C: centrifugal (14,000g, 4 ℃, 20min), collect supernatant, to filter, filtrating adds 12% (W/V) PEG-6000, centrifugal (14,000g, 4 ℃, 20min);
D: abandoning supernatant, deposition use the PBS (pH7.4) of 10ml to suspend, and add 12% (W/V) PEG-6000, centrifugal (14,000g, 4 ℃, 20min);
E: after deposition is used 1.2ml PBS dissolving, use dialysis band (interception 80000-140000) to dialyse, PBS (pH7.4) dialysis 3 times.
The external neutralizing effect that 2 anti-VACA-IgY infect VACA
Different dilution anti-VACA-IgY antibody mix with the viral suspension of equivalent, and mixture joins in the Vero E6-7 cell suspension and cultivates, and after the formalin fixed, NBB dyeing is calculated in the spot and titre (PRNT
50).
The method of 3 anti-VACA-IgY antibody coupling immunomagnetic beadses
The anti-VACA-IgY antibody (1: 100,000) and the magnetic bead (Dynabeads of the high titre of purifying
TMM-280) hatch on shaking table according to the ratio of optimizing, after the washing, 37 ℃ of sealings down.
Description of drawings
Fig. 1 is to use indirect immunofluorescence (indirect immunoflourence assay) to measure anti-VACA-IgY antibody: wherein, and (a) for infecting the Hep2 cell of VACA virus; The Hep2 cell (b) that anti-VACA-IgY (1: 1000) combines VACA to infect is after the Hep2 cell of anti-VACA-IgY (1: 1000) and VACA infection is hatched, observed photo; Infecting VACA virus Hep2 cell (negative control) Fig. 2 is the antibody activity that Western blot analyzes anti-VACA-IgY; Fig. 3 is that the anti-VACA-IgY of immune electron microscopy combines virus particle: a.VACA virus particle and unconjugated anti-and two anti-, and the b.VACA virus particle is anti-with gold mark goat-anti chicken IgG two, and c.VACA particle and IgY antibody (1: 1000) are marked goat-anti chicken IgG two with gold and are resisted.
Embodiment below in conjunction with accompanying drawing and contriver provide is done further detailed description to the present invention.
Embodiment:
(1) specific anti VACA-IgY antibody of the present invention can prepare through following method:
The antigenic preparation of 1VACA
A) VACA many times inoculation 11 age in days chick embryo fibroblasts (CEF), hatch 4 days after, ultracentrifugation in the sucrose is suspended from the PBS damping fluid;
B) spot measuring VACA titre after use Superlysoform carries out deactivation, is stored-70 ℃;
2 immunization methods
Initial immunity is with Freund emulsive antigen intramuscular injection immunity white Leghorn in 16 age in week, immunizing dose 400 μ g; Just exempt to exempt from back 36 days two Shi Yongfeishi Freund, immunizing dose 400 μ g; Three exempt from after 72 days, immunizing dose 2000 μ g; Exempted from immunizing dose 1000 μ g in 148 days four; Exempted from immunizing dose 2000 μ g in 319 days five; Exempted from immunizing dose 2000 μ g in 389 days six.Collect egg every day, make marks, 4 ℃ of preservations.
The purifying of 3IgY
The separation of IgY and purifying adopt polyethylene glycol precipitation, and method is following:
A): separate yolk and egg white, collect yolk, use PBS (pH7.4) to dilute in yolk according to 1: 2;
B): add the PEG-6000 of 3.5% (W/V), fully stir room temperature effect 10min on the shaking table;
C): centrifugal (14,000g, 4 ℃, 20min), collect supernatant, to filter, filtrating adds 12% (W/V) PEG-6000, centrifugal (14,000g, 4 ℃, 20min);
D): abandoning supernatant, deposition use the PBS (pH7.4) of 10ml to suspend, and add 12% (W/V) PEG-6000, centrifugal (14,000g, 4 ℃, 20min);
E): after deposition is used 1.2ml PBS dissolving, use dialysis band (interception 80000-140000) to dialyse, PBS (pH7.4) dialysis 3 times.
(2) the present invention is through above-mentioned antigen prepd and immune programme for children, and through separating and purifying, obtains the anti-VACA-IgY antibody of high specific, and in can be used as and medicine, treatment VACA infects, and its therapeutic action sharp experimental implementation in external carrying out is following:
Different dilution anti-VACA-IgY antibody are entreated liquid with the virus of equivalent and are mixed, and mixture joins in the Vero E6-7 cell suspension and cultivates, and formalin fixed after the dyeing, is calculated in the spot and titre (PRNT
50).
(3) method of anti-VACA-IgY antibody coupling immunomagnetic beads is used for VACA antigen is carried out enrichment, is used to detect VACA.
1 anti-VACA-IgY antibody coupling immunomagnetic beads
The anti-VACA-IgY antibody and the magnetic bead (Dynabeads of the high titre of purifying
TMM-280) hatch on shaking table according to the ratio of optimizing, after the washing, sealing.
2 immunomagnetic beads enrichment VACA antigens
The magnetic bead that lotus root connects anti-VACA-IgY antibody joins the VACA virus of dilution, after hatching, collects magnetic bead; After the washing, use DNA extraction test kit (InstaGen matrix, Bio-Rad; USA) viral DNA of release enrichment; Real-time quantitative PCR (real-time PCR assay) is measured its enriching quantity, calculate recovery rate.
Embodiment 1: the preparation of anti-VACA-IgY antibody
1) the antigenic preparation of VACA:
A:VACA many times inoculation 11 age in days chick embryo fibroblasts (CEF), hatch 4 days after, ultracentrifugation in the sucrose is suspended from the PBS damping fluid;
B: spot measuring VACA titre, after the Superlysoform deactivation, store-70 ℃;
2) immunization method:
Initial immunity is with Freund emulsive antigen intramuscular injection immunity white Leghorn in 16 age in week, immunizing dose 400 μ g; Just exempt to exempt from back 36 days two Shi Yongfeishi Freund, immunizing dose 400 μ g; Three exempt from after 72 days, immunizing dose 2000 μ g; Exempted from immunizing dose 1000 μ g in 148 days four; Exempted from immunizing dose 2000 μ g in 319 days five; Exempted from immunizing dose 2000 μ g in 389 days six.Collect egg every day, make marks, 4 ℃ of preservations.
3) purifying of IgY
The separation of IgY and purifying adopt polyethylene glycol precipitation, and method is following:
A: separate yolk and egg white, collect yolk, use PBS (pH7.4) to dilute in yolk according to 1: 2;
B: add the PEG-6000 of 3.5% (W/V), fully stir room temperature effect 10min on the shaking table;
C: centrifugal (14,000g, 4 ℃, 20min), collect supernatant, to filter, filtrating adds 12% (W/V) PEG-6000, centrifugal (14,000g, 4 ℃, 20min);
D: abandoning supernatant, deposition use the PBS (pH7.4) of 10ml to suspend, and add 12% (W/V) PEG-6000, centrifugal (14,000g, 4 ℃, 20min);
E: after deposition is used 1.2ml PBS dissolving, use dialysis band (interception 80000-140000) to dialyse, PBS (pH7.4) dialysis 3 times.
Embodiment 2: anti-VACA-IgY antibody activity detects
1) mensuration of IgY antibody titer
IIF is measured anti-VACA antibody titer, and method is following:
A:12 porocyte culture plate is cultivated the Hep2 cell, and inoculated with VA CA virus is cultivated;
B: acetone fixed adds the anti-VACA-IgY that dilutes, 5%CO
2(37 ℃) are cultivated 1h down;
C:PBS washed twice, distilled water wash once add the anti-chicken IgY of the donkey antibody of FITC mark after the drying, hatch 1h;
D: fluorescence microscope, the Hep2 cell that does not infect is as negative control
The result shows that anti-VACA-IgY begins to produce antibody after just exempting from for 2 weeks, behind first time booster immunization, i.e. and the 6th week; Antibody titers reaches 1: 10, in 000 (21 week) behind the booster immunization for the third time, reaches peak 1: 100,000; At 67-377 days, antibody titers maintained 1: 10, more than 000
2) among the anti-VACA with active mensuration
50% spot reduces neutralization test (PRNT
50) measure IgY antibody neutralization active: VACA virus is hatched with the anti-VACA-IgY that equivalent is diluted; Mixture joins in the VeroE6-7 enchylema and hatches; Add CMC 99.5, after hatching, fix, dye.The result shows, anti-VACA-IgY is behind second time booster immunization, and NAT reached peak 1: 320; And continue to booster immunization for the third time, descend then, behind the 4th time and the 5th booster immunization; NAT continues to reduce, and at the 373rd day, does not detect NAT.
Embodiment 3: anti-VACA-IgY western blot analyzes
The VACA virus particle is carried out the SDS-PAGE electrophoresis, transfer to nitrocellulose membrane after, seal 1h under the room temperature; Add after anti-VACA-IgY hatches, it is anti-to add the anti-chicken of rabbit two, after the washing; Add the substrate colour developing, the anti-VACA-IgY of result respectively with virus particle 100KDa and 55KDa particle reaction.
Embodiment 4: anti-VACA-IgY immune electron microscopy
Copper particle and VACA virus solution are hatched 5min, after the washing, add different dilution anti-VACA-IgY, after the washing, add goat-anti chicken two anti-(1: 100), use the projection electron microscopic observation.The result shows that anti-VACA-IgY can be attached to the VACA particle surface.
Claims (2)
1. anti-vaccinia virus VACA (Vaccinia virus; VACA) preparation of yolk antibody IgY; It is characterized in that: said anti-VACA yolk antibody through the spot immune adsorption experiment (Dot ImmunobindingAssay, DIBA) proof can with VACA antigen generation specificity association reaction; (immunoelectron microscopy IEM) observes immuno-electron microscope, and the anti-VACA yolk antibody of gold mark can specificity combine VACA virus particle surface; (western blot WB) analyzes the immunity marking, and specificity combines vaccinia virus but yolk antibody is at the 100KDa place, presents specific stain; (Indirect immunofluorescence IFA) measures titre to indirect immunofluorescence, and yolk antibody and reached the highest in the 21st week behind the 3rd booster immunization; 50% spot neutralization test (plaque reduction neutralization test, PRNT
50) show that behind the booster immunization, it is maximum that NAT reaches for the second time; When antibody titers reaches the highest, IgY is attached to magnetic bead, concentrates the vaccinia virus of different extension rates, spissated virus quantity uses real-time fluorescence PCR to detect its content, and when viral level reached 5PFU/ml, its recovery reached 35.4%.
2. one kind prepares anti-vaccinia virus yolk antibody, and the method for promptly anti--VACA IgY is characterized in that adopting following process step:
(1) the antigenic preparation of VACA
A): VACA many times inoculation 11 age in days chick embryo fibroblasts (CEF), hatch 4 days after, ultracentrifugation is suspended from the PBS damping fluid;
B): spot measuring VACA titre, the Superlysoform deactivation, store-70 ℃ subsequent use.
(2) immunization method
Initial immunity is with Freund emulsive antigen intramuscular injection immunity white Leghorn in 16 age in week, immunizing dose 400 μ g; Just exempt to exempt from back 36 days two Shi Yongfeishi Freund, immunizing dose 400 μ g; Three exempt from after 72 days, immunizing dose 2000 μ g; Exempted from immunizing dose 1000 μ g in 148 days four; Exempted from immunizing dose 2000 μ g in 319 days five; Exempted from 389 days six, immunizing dose 2000 μ g collect egg every day, make marks 4 ℃ of preservations.
(3) purifying of IgY
The separation of IgY and purifying adopt polyethylene glycol precipitation, and method is following:
A): separate yolk and egg white, collect yolk, use PBS (pH7.4) to dilute in yolk according to 1: 2;
B): add the PEG-6000 of 3.5% (W/V), fully stir room temperature effect 10min on the shaking table;
C): centrifugal (14,000g, 4 ℃, 20min), collect supernatant, to filter, filtrating adds 12% (W/V) PEG-6000, centrifugal (14,000g, 4 ℃, 20min);
D): abandoning supernatant, deposition use the PBS (pH7.4) of 10ml to suspend, and add 12% (W/V) PEG-6000, centrifugal (14,000g, 4 ℃, 20min);
E): after deposition is used 1.2ml PBS dissolving, use dialysis band (interception 80000-140000) to dialyse, PBS (pH7.4) dialysis 3 times.
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| CN104341499A (en) * | 2014-11-06 | 2015-02-11 | 遵义医学院 | Preparation process of tetravalent yolk antibody preparation for resisting measles and bronchocephalitis, diphtheritis and lockjaw |
| CN104356232A (en) * | 2014-10-27 | 2015-02-18 | 西北农林科技大学 | Preparation method of specific IgY for resisting bovine viral diarrhea virus protein E2 |
| CN104725502A (en) * | 2015-02-04 | 2015-06-24 | 陕西溯源农业发展有限公司 | Preparation method of anti-small ruminant animal specificity IgY |
| CN110669128A (en) * | 2019-09-30 | 2020-01-10 | 南京道大药业有限公司 | Preparation method and application of anti-herpes simplex virus composite IgY antibody |
| CN111303277A (en) * | 2020-02-19 | 2020-06-19 | 中国人民解放军军事科学院军事医学研究院 | An immunoglobulin F (ab') for resisting smallpox virus2And method for preparing the same |
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| CN104356232A (en) * | 2014-10-27 | 2015-02-18 | 西北农林科技大学 | Preparation method of specific IgY for resisting bovine viral diarrhea virus protein E2 |
| CN104341499A (en) * | 2014-11-06 | 2015-02-11 | 遵义医学院 | Preparation process of tetravalent yolk antibody preparation for resisting measles and bronchocephalitis, diphtheritis and lockjaw |
| CN104725502A (en) * | 2015-02-04 | 2015-06-24 | 陕西溯源农业发展有限公司 | Preparation method of anti-small ruminant animal specificity IgY |
| CN110669128A (en) * | 2019-09-30 | 2020-01-10 | 南京道大药业有限公司 | Preparation method and application of anti-herpes simplex virus composite IgY antibody |
| CN110669128B (en) * | 2019-09-30 | 2022-11-29 | 南京道大药业有限公司 | Preparation method and application of anti-herpes simplex virus composite IgY antibody |
| CN111303277A (en) * | 2020-02-19 | 2020-06-19 | 中国人民解放军军事科学院军事医学研究院 | An immunoglobulin F (ab') for resisting smallpox virus2And method for preparing the same |
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