CN108484757A - A kind of pseudorabies virus antiserum and preparation method thereof - Google Patents
A kind of pseudorabies virus antiserum and preparation method thereof Download PDFInfo
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- CN108484757A CN108484757A CN201810377114.6A CN201810377114A CN108484757A CN 108484757 A CN108484757 A CN 108484757A CN 201810377114 A CN201810377114 A CN 201810377114A CN 108484757 A CN108484757 A CN 108484757A
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- pseudorabies
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
- C07K16/085—Herpetoviridae, e.g. pseudorabies virus, Epstein-Barr virus
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
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Abstract
The present invention relates to a kind of pseudoabies disease virus antiseras and preparation method thereof.The present invention is directed to provide the pseudorabies virus positive serum met the requirements for the monitoring of pseudoabies diagnostic techniques, diagnostic reagent and porcine pseudorabies vaccine quality.Pseudorabies virus positive serum specificity according to the present invention is high, and neutralization titer is up to 1:2000 times or more, the exogenous virus that can meet pseudorabies living vaccines is examined and diagnostic test；In addition, animal is immunized in the invention and uses the healthy goat without containing pig source common causative, is easy to control on the degree of purity of experimental animal, immunogene is pseudoabies inactivated vaccine and live vaccine, it is easy to get and avoids being immunized using the strong poison of pseudorabies virus, reduce bio-safety risk；Merely through five immune antiserums that can obtain high-titer, method is simple, is suitble to extensive prepare.
Description
Technical field
The present invention relates to a kind of pseudorabies virus antiserums and preparation method thereof, belong to veterinary biologics technology neck
Domain.
Background technology
Pseudoabies (Aujeszkys disease) is by I type of Alphaherpesvirinae herpesvirus suis in herpetoviridae
A kind of caused acute infectious disease is current one of most serious infectious disease in the world, and is widely current in China, is had resulted in tight
Weight economic loss.Pseudo- mad dog diagnostic techniques, diagnostic reagent and vaccine etc. are the key that the anti-systems of the disease.The diagnostic techniques of the disease and
Vaccine quality monitoring is required for pseudorabies virus positive serum, but current have not been reported can meet the pseudorabies virus used
The preparation method of hyper-immune serum.The present invention is for the first time immunized goat with pseudorabies living vaccines and inactivated vaccine, is had
There is the specific positive serum of high neutralization titer, and make the pure property of serum more easy to control and reliable, meets pseudoabies
The exogenous virus of live vaccine is examined and diagnostic test.
Invention content
The purpose of the present invention：A kind of tool for needed for the inspection of pseudorabies living vaccines exogenous virus and diagnostic test is provided
There is the specific pseudorabies virus antiserum of high neutralization titer.
Technical scheme of the present invention
1. a kind of pseudorabies virus antiserum, it is characterised in that the pseudorabies virus antiserum is normal using no swine disease
See that the goat of pathogen is prepared, and the antiserum neutralization titer is up to 1:2000 times or more, it can be used for pseudoabies work epidemic disease
The exogenous virus of seedling is examined and diagnostic test.
2. a kind of sero-fast preparation method of pseudorabies virus, it is characterised in that the sero-fast preparation method is：
(1) experimental animal：For the goat without swine disease common causative；
(2) immunogene：Using the pseudoabies inactivated vaccine and live vaccine of commercialization；
(3) preparation of hyper-immune serum：Examine qualified experiment with goat with pseudoabies inactivated vaccine and live vaccine altogether into
Row is repeatedly immune, the pseudo- Rabies virus antibody potency of detection before being immunized every time, and last time is immunized latter all serum neutralize antibody titers and reaches
It is taken a blood sample to peak value using arteria carotis to immune sheep lethal, detaches serum；
(4) inspection of hyper-immune serum：
1) physical behavior, steriling test, mycoplasma are examined：Meet the requirements；
2) titration：The neutralization titer that hyper-immune serum is measured with neutralization test and ELISA method, up to 1:2000 or more；
3) specific assay：Detect aftosa, swine fever, porcine reproductive and respiratory syndrome, porcine parvovirus, pig circular ring virus 2
Viral disease, pseudoabies and bovine viral diarrhoea/mucosal disease antibody, are feminine gender；
4) exogenous virus is examined：No exogenous virus pollution.
The specific implementation mode of the present invention
1. the preparation of pseudorabies virus specific antisera
(1) experimental animal：Or so 6 monthly ages, not not immune aftosa goat, BVDV, pseudoabies, cloth Lu Shi
Bacterium disease, blue tongue disease, peste des petits ruminants and sheep pox antibody are feminine gender.
(2) immunogene：Pseudoabies inactivated vaccine and pseudorabies living vaccines
(3) immune programme：Multi-time no is carried out with pseudoabies inactivated vaccine and pseudorabies living vaccines with goat to testing
Epidemic disease.
(4) prepared by serum：The situation of change of antibody level after latter week blood sampling monitoring is immunized is immunized every time.Last time is exempted from
The latter all antibody levels of epidemic disease reach highest, and arteria carotis blood sampling is lethal, detaches serum.- 20 DEG C of freezen protectives, it is spare.
2. the sero-fast inspection of pseudorabies virus
1. steriling test：According to Republic of China Veterinary Pharmacopoeia (the Chinese veterinary pharmacopoeia committee, People's Republic of China (PRC) beast
Pharmacopeia, two 〇 First Five-Year Plans year version three, Chinese agriculture publishing house, 2016, hereinafter referred to as《Chinese veterinary pharmacopoeia》) as defined in method carry out nothing
Bacterium is examined or purely examines, and asepsis growth is answered.
2. mycoplasma is examined：According to《Chinese veterinary pharmacopoeia》Defined method carries out mycoplasma inspection, should be grown without mycoplasma.
3. neutralization titer measures
According to《Chinese veterinary pharmacopoeia》Defined method carries out in neutralization test measurement serum and 200TCID50Pseudoabies disease
Malicious Bartha plants of neutralization titer,
4. specific assay：Detect aftosa, swine fever, porcine reproductive and respiratory syndrome, porcine parvovirus, pig circular ring virus 2
Viral disease and bovine viral diarrhoea/mucosal disease antibody should be negative, and pseudorabies virus antibody is the positive；
5. exogenous virus is examined：It should be polluted without exogenous virus.
The positive effect of the present invention
The present invention relates to a kind of pseudoabies disease virus antiseras and preparation method thereof.The present invention is directed to be examined for pseudoabies
Disconnected technology, diagnostic reagent and the monitoring of porcine pseudorabies vaccine quality provide the pseudorabies virus positive serum met the requirements.
Pseudorabies virus positive serum specificity according to the present invention is high, and neutralization titer is up to 1:2000 times or more, it can meet
The exogenous virus of pseudorabies living vaccines is examined and diagnostic test；In addition, animal is immunized in the invention using normal without containing pig source
The healthy goat for seeing pathogen, is easy to control on the degree of purity of experimental animal, and immunogene is pseudoabies inactivated vaccine and work
Vaccine is easy to get and avoids being immunized using the strong poison of pseudorabies virus, reduces bio-safety risk；Merely through five times
The immune antiserum that can obtain high-titer, method is simple, is suitble to extensive prepare.Pseudorabies virus pseudoabies
The live vaccine present invention improves this to solve the problems in pseudoabies diagnostic techniques, diagnostic reagent and vaccine quality monitoring
The inspection work of industry, while escorting for the promotion of the industry products quality and the safety of aquaculture.Pseudorabies virus
Pseudorabies virus
Embodiment
Following embodiment is to better illustrate the present invention, is not limited the invention.
Embodiment 1
--- the preparation of pseudorabies virus specific antisera
1. experimental animal：Or so 6 monthly ages, the goat for being not suffering from not immune aftosa, and BVDV, pseudo- mad dog after testing
Disease, brucellosis, blue tongue disease, peste des petits ruminants and sheep pox antibody are feminine gender.
2. immunogene：Pseudoabies inactivated vaccine and pseudorabies living vaccines
3. immune programme：
Qualified experiment is examined repeatedly to be immunized with pseudoabies inactivated vaccine and pseudorabies living vaccines with goat.
4. prepared by serum
The situation of change of antibody level after latter week blood sampling monitoring is immunized is immunized every time.Latter Zhou Kangti is immunized in last time
Level reaches highest, and arteria carotis blood sampling is lethal, detaches serum.- 20 DEG C of freezen protectives, it is spare.
Embodiment 2
--- the sero-fast inspection of pseudorabies virus
1. steriling test：According to Republic of China Veterinary Pharmacopoeia (the Chinese veterinary pharmacopoeia committee, People's Republic of China (PRC) beast
Pharmacopeia, two 〇 First Five-Year Plans year version (three), Chinese agriculture publishing house, 2016, hereinafter referred to as《Chinese veterinary pharmacopoeia》) as defined in method carry out
Steriling test is purely examined, asepsis growth.
2. mycoplasma is examined：According to《Chinese veterinary pharmacopoeia》Defined method carries out mycoplasma inspection, no mycoplasma growth.
3. neutralization titer measures
According to《Chinese veterinary pharmacopoeia》Defined method carries out in neutralization test measurement serum and 200TCID50Pseudoabies disease
Malicious Bartha plants of neutralization titer, test result are shown in Table 1.
Pseudorabies virus Specific serum antibodies level monitoring result prepared by 1 present invention of table
| Number of animals | 1 | 2 | 3 | 4 |
| Before head exempts from | 0 | 0 | 0 | 0 |
| Before second immune | 1:4 | 1:4 | 1:16 | 1:8 |
| Before third time is immune | 1:32 | 1:16 | 1:64 | 1:64 |
| Before 4th time immune | 1:128 | 1:64 | 1:256 | 1:256 |
| Before 5th time immune | 1:1024 | 1:256 | 1:1024 | 1:1024 |
| Before 6th time immune | 1:5623 | 1:2506 | 1:8192 | 1:2818 |
Test result shows that pseudorabies virus antibody neutralization titer after the 5th is immune reaches peak value, suitable at this moment
Between largely taken a blood sample.
4. specific assay：Detect aftosa, swine fever, porcine reproductive and respiratory syndrome, porcine parvovirus, pig circular ring virus 2
Viral disease and bovine viral diarrhoea/mucosal disease antibody, are feminine gender, and pseudoabies antibody is the positive；
5. exogenous virus is examined：No exogenous virus pollution.
Embodiment 3
--- pseudorabies virus specific serum is examined for pseudorabies living vaccines diagnostic test and exogenous virus
1. pseudorabies living vaccines (Bartha-K61 plants), viral level 106.0TCID50/ head part.
2. test method：
According to《Chinese veterinary pharmacopoeia》With《Pseudorabies living vaccines》Quality standard (CVP3/2015/HYM/029) is differentiated
It examines and exogenous virus is examined.
3. diagnostic test：
To determine that the serum can be used for the diagnostic tests of pseudorabies living vaccines, vaccine is diluted to 2.0 ×
102.0TCID50The vaccine diluent of/0.1mL, then carried out with solution after 4 times of dilutions of PBS (1/15mol/L, pH7.2) with the serum
Mixed in equal amounts, after neutralizing 90min at 37 DEG C, inoculating cell sets 37 DEG C of cultures.The control group for not neutralizing vaccine is set simultaneously, sets same item
It is cultivated under part.Observation 7 days judges result.Characteristic cytopathy should occur in cellular control unit, and neutralization group cell is answered acellular
Lesion.
Test result shows that the serum can be used for the diagnostic test of pseudorabies living vaccines twice.
4. exogenous virus is examined
To determine that the serum can be used for the exogenous virus inspection of pseudorabies living vaccines, according to current edition《Chinese veterinary pharmacopoeia》
Three progress exogenous virus inspections, by 1ml serum and 1 part pseudorabies living vaccines (Bartha-K61 plants) (viral level
106.0TCID50/ head part) mixing after, is neutralized 1 hour at 37 DEG C, be respectively adopted Vero cells, MDBK cells and PK-15 cells biography
3 cultures of generation.Per generation observation 7 days, judges result.Neutralization group cell answers acellular lesion.
Test result shows the pseudorabies living vaccines (Bartha-K61 plants) of 1ml serum energy 1 part of complete neutralization twice
(viral level 106.0TCID50/ head part).
5. conclusion
Pseudorabies virus specific antisera can be used for pseudorabies living vaccines (Bartha-K61 plants) diagnostic test and
Exogenous virus is examined.
Claims (2)
1. a kind of pseudorabies virus antiserum, it is characterised in that the pseudorabies virus hyper-immune serum be using goat prepare and
At；And the neutralization titer of the hyper-immune serum is up to 1:2000 times or more, it can be used for the exogenous virus of pseudorabies virus live vaccine
Inspection and diagnostic test.
2. a kind of sero-fast preparation method of Pseudorabies virus, it is characterised in that：
(1) experimental animal：For the healthy goat without swine disease common causative；
(2) immunogene：Swine pseudorabies vaccine and pseudorabies disease live-vaccine；
(3) preparation of hyper-immune serum：Qualified experiment is examined to be lived with swine pseudorabies vaccine and porcine pseudorabies with goat
Vaccine is immunized, and immune preceding using the pseudo- Rabies virus antibody potency of ELISA method detection every time, latter all serum is immunized in last time
Neutralize antibody titers reach peak value, lethal, the sterile separation serum of arteria carotis blood sampling；
(4) inspection of hyper-immune serum：
1) it is qualified to be examined through physical behavior, steriling test, mycoplasma；
2) titration：Sero-fast neutralization titer is measured with neutralization test；
3) specific assay：Detect aftosa, swine fever, porcine reproductive and respiratory syndrome, porcine parvovirus, Porcine circovirus desease
It is feminine gender with the antibody such as bovine viral diarrhoea/mucosal disease, pseudoabies antibody is the positive；
4) exogenous virus is examined：No exogenous virus pollution.
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| CN201810377114.6A CN108484757A (en) | 2018-04-25 | 2018-04-25 | A kind of pseudorabies virus antiserum and preparation method thereof |
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| CN201810377114.6A CN108484757A (en) | 2018-04-25 | 2018-04-25 | A kind of pseudorabies virus antiserum and preparation method thereof |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5122447A (en) * | 1989-01-23 | 1992-06-16 | Iowa State University Research Foundation, Inc. | Method of detecting pseudorabies virus specific serum antibody by use of a universal diagnostic antigen |
| CN101862452A (en) * | 2010-07-22 | 2010-10-20 | 山西芮城亚宝兽药有限公司 | Method for preparing swine multi-valent antiserum compound preparation |
| CN102353783A (en) * | 2011-06-29 | 2012-02-15 | 武汉科前动物生物制品有限责任公司 | Kit for detecting pig pseudorabies virus antibodies and block enzyme-linked immuno sorbent assay (ELISA) method |
| CN105732807A (en) * | 2016-02-24 | 2016-07-06 | 福州大北农生物技术有限公司 | Porcine pseudorabies virus positive serum preparation method |
-
2018
- 2018-04-25 CN CN201810377114.6A patent/CN108484757A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5122447A (en) * | 1989-01-23 | 1992-06-16 | Iowa State University Research Foundation, Inc. | Method of detecting pseudorabies virus specific serum antibody by use of a universal diagnostic antigen |
| CN101862452A (en) * | 2010-07-22 | 2010-10-20 | 山西芮城亚宝兽药有限公司 | Method for preparing swine multi-valent antiserum compound preparation |
| CN102353783A (en) * | 2011-06-29 | 2012-02-15 | 武汉科前动物生物制品有限责任公司 | Kit for detecting pig pseudorabies virus antibodies and block enzyme-linked immuno sorbent assay (ELISA) method |
| CN105732807A (en) * | 2016-02-24 | 2016-07-06 | 福州大北农生物技术有限公司 | Porcine pseudorabies virus positive serum preparation method |
Non-Patent Citations (4)
| Title |
|---|
| H J NAUWYNCK等: "Effect of specific antibodies on the cell-associated spread of pseudorabies virus in monolayers of different cell types", 《ARCH VIROL》 * |
| 孟日增等: "猪伪狂犬病病毒多克隆抗体的制备及免疫学活性研究", 《中国畜牧兽医》 * |
| 王玉玲: "猪伪狂犬病病毒高免血清的制备及应用", 《福建畜牧兽医》 * |
| 秦刚: "不同免疫程序对制备狂犬病抗血清的免疫效果比较", 《山东畜牧兽医》 * |
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Application publication date: 20180904 |