CN106771186B - A kind of antibody test method of hog cholera genetic engineering subunit vaccine effect - Google Patents
A kind of antibody test method of hog cholera genetic engineering subunit vaccine effect Download PDFInfo
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- CN106771186B CN106771186B CN201611105695.5A CN201611105695A CN106771186B CN 106771186 B CN106771186 B CN 106771186B CN 201611105695 A CN201611105695 A CN 201611105695A CN 106771186 B CN106771186 B CN 106771186B
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- 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
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
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- 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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
Abstract
The present invention provides a kind of swine fever virus genetic engineering subunit vaccines, efficacy test method, take the susceptible rabbit of health as checked object, are divided into control group and immune group；Immune group rabbit is injected, swine fever virus genetic engineering subunit vaccine is injected, and the rabbit of control group does not inject any substance；After vaccinating 21, Culling heart blood is carried out to the rabbit of immune group and control group, cardiac blood takes supernatant after centrifugation respectively, detects swine fever ELSIA antibody respectively, and antibody at least 4/5 is that the positive then proves that swine fever virus genetic engineering subunit vaccine efficacy test is qualified.Swine fever virus genetic engineering subunit vaccine efficacy test method of the invention, the alternative efficacy test that Immunization is carried out with pig, due to examining animal rabbit easily to obtain, save inspection cost, round of visits is short, is not related to swine fever strong virus attack, biological safety height.
Description
Technical field
The present invention relates to a kind of antibody test methods of hog cholera genetic engineering subunit vaccine (293T-E2) effect, belong to
Veterinary biologics field.
Background technique
Swine fever (slassical swine fever, CFS), for main feature, is in acute or chronic warp with bleeding and fever
It crosses, is a kind of pair of great infectious disease of pig harmfulness, A class zoonosis is classified as by International Office of Epizootics.In world many countries
And area, traditional vaccination are the important means for controlling swine fever.Attenuated vaccine used at present is mainly Chinese swine fever rabbitization
The Thireval vaccine of attenuated vaccine, Japan's GPE vaccine and France.The popular form in later period the 1970s, swine fever occurs
Great changes.Sporadic popular and non-typical swine fever symptom the generation in area, especially recent domestic application monoclonal
Discovery has a different reaction patterns when antibody is to C- plants of detections, and hog cholera immune unsuccessfully etc., shows the antigenicity of swine fever virus repeatedly
There may be variations, so that someone be made to propose query to traditional attenuated vaccine.With the increase of passage number and to difference
The adaptation sexual reproduction of cell, vaccine strain may occur heterogeneous or antigenic drift in addition for decades CSFV in mass immunization
Antigenic variation is likely to occur under inoculation pressure.
These situations all show that existing traditional vaccine has not adapted to the needs of new situations, research and develop Novel pig
Pestilence seedling machine is trend of the times.The rise and development of Protocols in Molecular Biology are established for the research and development of New Kind of Vaccine for Classical Swine Fever
Basis.Gene delection attenuated vaccine, subunit vaccine and live vector vaccine are the hot spots studied at present.
Swine fever virus genetic engineering subunit vaccine belongs to inactivated vaccine, except using target animals (pig) progress Immunization
Outside efficacy test method, there is presently no available alternatives, the method due to carrying out Immunization using target animals (pig)
Detection cycle is long (51 days), and raising and inspection cost are high, virulent using swine fever involved in checkout procedure, in order to solve the above problem,
It is badly in need of the efficacy test for establishing feasible effective experimental animal efficacy test method for inactivated vaccine, detection week can be shortened
Phase saves testing cost, improves bio-safety.
Summary of the invention
It is alternative normal the object of the present invention is to provide a kind of swine fever virus genetic engineering subunit vaccine efficacy test method
Rule carry out the efficacy test of Immunization with pig, to make up the blank of existing subunit vaccine efficacy test.
The method of inspection of the present invention, includes the following steps, namely：
1) it takes the susceptible rabbit of health as checked object, is randomly divided into control group and immune group according to weight, gender, exempts from
Epidemic disease group is at least provided with 2 parallel groups；
It is negative rabbit that the susceptible rabbit, which is swine fever virus ELISA antibody,；
2) swine fever virus genetic engineering subunit vaccine (293T-E2) to be detected is injected to immune group rabbit, and compareed
The rabbit of group is not injected；The rabbit of the rabbit and control group that vaccinate is raised according to normal feeding manner；
3) after step 2) vaccinates 21, Culling heart blood, blood warp are carried out to the rabbit of immune group and control group
Supernatant is taken after crossing centrifugation, detects swine fever ELSIA antibody respectively, antibody is that the positive then proves hog cholera genetic engineering subunit vaccine
Reach protective effect.
Preferably, in step 1), the control group does not set parallel group；The immune group is equipped with parallel group, Mei Geping
The rabbit number of elements that row group is set up is identical as control group.
Preferably, in step 2), the injection system of the swine fever virus genetic engineering subunit vaccine is venae subcutaneae
Injection, injection site are the neck of rabbit.
Preferably, in step 2), the injection dosage of the swine fever virus genetic engineering subunit vaccine is 0.2ml/.
Preferably, in step 3), speed when centrifugation is 7000r/min, and centrifugation time is 10 minutes.
Determine according to the method for the present invention, control group rabbit swine fever ELSIA antibody is feminine gender, and the rabbit of immune group, pig
Pest ELSIA antibody is that positive ratio is at least 80%.Swine fever virus genetic engineering subunit vaccine efficacy test of the invention
Method, the alternative efficacy test that Immunization is carried out with pig save inspection cost, inspection due to examining animal rabbit easily to obtain
It is short to test the period, is not related to swine fever strong virus attack, and biological safety is high.
Specific embodiment
The present invention is using the susceptible rabbit immunization swine fever virus genetic engineering subunit vaccine (293T-E2) of health, after being immunized
Blood sampling detection swine fever virus ELISA antibody, passes through the immune effect of antibody yin-yang sex determination vaccine.
The present invention is further explained in the light of specific embodiments.
Embodiment 1：A kind of hog cholera genetic engineering subunit vaccine (293T-E2) efficacy test method
Include the following steps：
(1) it takes 20 healthy susceptible rabbit (swine fever virus ELISA antibody is negative rabbit) as checked object, presses
4 groups, every group 5 are randomly divided into according to weight, gender；3 groups are immune group, and 1 group is control group；
(2) immune group rabbit is injected, 3 groups of hog cholera genetic engineerings for injecting 0.1ml, 0.2ml, 0.5ml respectively are sub-
Subunit vaccine (293T-E2), and the rabbit of control group does not inject any substance；The rabbit of the rabbit and control group that vaccinate by
It is raised according to normal feeding manner；
(3) after step (2) vaccinates 21, Culling heart blood, cardiac blood are carried out to the rabbit of immune group and control group
Supernatant is taken after being centrifuged 10min respectively with 7000r/min, detects swine fever ELSIA antibody respectively (according to the production of IDEXX company
Antibody against swine fever virus detection kit specification is operated, and detection time is 4 hours), antibody is that the positive then proves swine fever base
Because engineering subunit vaccine (293T-E2) reaches protective effect.The results are shown in Table 1：
Table 1：Antibody test
Note：Blocking rate >=40% is positive (+)；30% < blocking rate < 40% is suspicious (±)；Blocking rate≤30% is
Negative (-).
As shown in Table 1, after rabbit injection hog cholera genetic engineering subunit vaccine (293T-E2), specificity can be generated
Antibody, with 0.1ml/ dose immunization, antibody blocking rate is between 10.3-71.0%, antibody positive rate 80%, with 0.2ml/
Only, 0.5ml/ dose immunization, for antibody blocking rate between 64.9-81.8%, antibody positive rate is 100%, and control group is anti-
For body blocking rate between 2.8-12.4%, antibody is feminine gender, it can be seen that hog cholera genetic engineering subunit vaccine (293T-E2)
Minimum immune dosage to rabbit is 0.1ml/, and when carrying out the efficacy test of vaccine, dosage is set to minimum immunizing agent
2 times of amount, i.e. 0.2ml/ is only.
Embodiment 2：Antibody test protects correlation detection with poison is attacked：
In embodiment 1, after the rabbit of immune group and control group carries out Culling heart blood, every rabbit carries out ear's intravenous injection
Live vaccines of hog cholera (spleen leaching source), 1.0ml/ is only；That injects live vaccines of hog cholera (spleen leaching source) measures a body temperature, note for first two days daily
It is primary to penetrate rear every 6 hours thermometrics, surveys 72 hours altogether；Injection Rabbits Before And After Temperature changing is judged to reaching protection when being no more than 1 DEG C
Effect.The results are shown in Table 2：
Table 2：Attack malicious protection
| Grouping | Animal species | Size of animal | Protective rate | Not protective rate |
| Immune group 1 (0.1ml/ is only) | Rabbit | 5 | 80% | 20% |
| Immune group 2 (0.5ml/ is only) | Rabbit | 5 | 100% | 0% |
| Immune group 3 (1.0ml/ is only) | Rabbit | 5 | 100% | 0% |
| Control group | Rabbit | 5 | 0% | 100% |
As shown in Table 2, rabbit injection hog cholera genetic engineering subunit vaccine (293T-E2) after, then with swine fever poison (swine fever spleen
Fever virus lapinized Chinese Strain in the live vaccine of leaching source) attack, it can achieve protective effect, in addition, antibody test has with poison protection is attacked
There is good correlation.
Embodiment 3：The method of inspection of the present invention is examined using and with target animals and is compared
Pig weight 1.5~3.0kg health susceptible (pestivirus ELISA antibody is feminine gender) rabbit 20 is taken, is randomly divided into 4
Group, every group 5,201401,201402,201403 batches of hog cholera genetic engineering subunit vaccines are subcutaneously injected in each neck of 1-3 group
(293T-E2), only, the 4th group is control group to 0.2ml/.21 days after exempting from, all Rabbit Heart blood samplings, 7000r/min is centrifuged 10 points
Clock draws supernatant and detects swine fever ELSIA antibody；Antibody is that the positive then proves hog cholera genetic engineering subunit vaccine (293T-E2)
Reach protective effect.The results are shown in Table 3：
Table 3：Antibody test
Note：Blocking rate >=40% is positive (+)；30% < blocking rate < 40% is suspicious (±)；Blocking rate≤30% is
Negative (-).
Susceptible (pestivirus PCR detects feminine gender, and ELISA antibody is feminine gender) the 30-60 age in days pig 20 of pig health is taken, it is random to divide
At 4 groups, every group 5,1-3 group is immune group, each musculi colli injects 1.0ml swine fever virus genetic engineering subunit vaccine, the 4th
Group is control group.28 days after exempting from, all pigs inject the virulent 1.0ml/ head of classical swine fever virus Shimen system.Attack after poison day thermometric twice, altogether
It surveys 16, the clinical manifestation of malicious pig is attacked in observation (eye conjunctivitis, appetite abolish, constipation and diarrhea are alternately present, spirit is depressed).Root
According to attacking before poison 2 and attacking thermometric and clinical observation comprehensive judgement after poison, determine when Temperature changing is no more than 1 DEG C and without clinical manifestation
To reach protective effect.The results are shown in Table 4：
Table 4：Attack malicious temperature of pig body reaction, clinical observation and dissect statistical form
Note：There is this symptom or has body temperature reaction in " √ " expression；"-" indicates to react without this symptom or without body temperature.
Table 6：Attack poison protection statistical form
| Grouping | Animal species | Size of animal | Protective rate | Not protective rate |
| Immune group 1 (1.0ml/) | 30-60 age in days pig | 5 | 100% | 0% |
| Immune group 2 (1.0ml/) | 30-60 age in days pig | 5 | 100% | 0% |
| Immune group 3 (1.0ml/) | 30-60 age in days pig | 5 | 100% | 0% |
| Control group | 30-60 age in days pig | 5 | 0% | 100% |
By table 3,4,5,6 it is found that carrying out antibody test with rabbit, 3 batches of vaccine immunity rabbit reach 5/5 antibody positive,
And carry out Immunization with pig the result shows that, 3 batches of vaccine immunity pigs it is equal 5/5 protection, it can be seen that, the inspection result of two methods
It is consistent, antibody test can be carried out with rabbit and can substitute to carry out Immunization with pig.
Examples detailed above is technical conception and technical characteristics to illustrate the invention, can not be limited with this of the invention
Protection scope.The equivalent transformation or modification that all essence according to the present invention is done, should all cover in protection scope of the present invention
Within.
Claims (4)
1. a kind of swine fever virus genetic engineering subunit vaccine efficacy test method, which is characterized in that the method includes such as
Under step：
1) it takes the susceptible rabbit of health as checked object, is randomly divided into control group and immune group, immune group according to weight, gender
At least provided with 2 parallel groups；
2) swine fever virus genetic engineering subunit vaccine to be detected is injected to immune group rabbit, and the rabbit of control group is not infused
It penetrates；The rabbit of the rabbit and control group that vaccinate is raised according to normal feeding manner；
3) after step 2) vaccinates 21, Culling heart blood carried out to the rabbit of immune group and control group, blood pass through from
Supernatant is taken after the heart, detects swine fever ELSIA antibody respectively, and antibody at least 4/5 is that the positive then proves that swine fever virus genetic engineering is sub-
Subunit vaccine efficacy test is qualified；
The susceptible rabbit is that swine fever virus ELISA antibody is negative rabbit；
The injection dosage of swine fever virus genetic engineering subunit vaccine in the step 2) is 0.2ml/；
It takes a blood sample within 21 days after injection in the step 2)；
The vaccine is 293T-E2 vaccine.
2. the method as described in claim 1, which is characterized in that the control group in the step 1) does not set parallel group；It is described
Immune group be equipped with parallel group, the rabbit number of elements that each parallel group is set up is identical as control group.
3. the method as described in claim 1, which is characterized in that in the step 2), the swine fever virus genetic engineering
The injection system of subunit vaccine is venae subcutaneae injection, and injection site is the neck of rabbit.
4. the method as described in claim 1, which is characterized in that the speed in the step 3) when centrifugation is 7000r/min,
Centrifugation time is 10 minutes.
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| CN104178505A (en) * | 2014-09-01 | 2014-12-03 | 华中农业大学 | Recombinant virus for expressing swine fever virus E2 gene, and preparation method and application thereof |
| CN105331636A (en) * | 2015-12-04 | 2016-02-17 | 广州伯尼兹生物科技有限公司 | Recombination cell line for stable expression of classical swine fever virus E2 and application thereof |
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| Development of a classical swine fever subunit marker vaccine and companion diagnostic test;RobJ.M.Moormann et al;《Veterinary Microbiology》;20001231;第73卷;第209-219页 * |
| Efficacy and stability of a subunit vaccine based on glycoprotein E2 of classical swine fever virus;A.Bouma et al;《Veterinary Microbiology》;19991231;第101-114页 * |
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