CN106645716A - Method for testing efficacy of porcine circovirus 2-type vaccine - Google Patents

Method for testing efficacy of porcine circovirus 2-type vaccine Download PDF

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CN106645716A
CN106645716A CN201611140272.7A CN201611140272A CN106645716A CN 106645716 A CN106645716 A CN 106645716A CN 201611140272 A CN201611140272 A CN 201611140272A CN 106645716 A CN106645716 A CN 106645716A
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porcine circovirus
small white
vaccine
white mouse
antibody horizontal
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张许科
孙进忠
乔荣岑
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Pulaike Biological Engineering Co Ltd
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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
    • 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
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Abstract

The invention belongs to the technical field of novel veterinary drug biological medicine and relates to a method for testing the efficacy of a porcine circovirus 2-type vaccine. The method comprises the following steps: immunizing a rat with a porcine circovirus 2-type inactivated vaccine, and then determining the specific antibody level of PCV2 in serum thereof, thereby evaluating the efficacy of the vaccine. The method for testing the effect of the porcine circovirus 2-type vaccine provided by the invention can be used for overcoming the difficulties of time-wasting and labor-wasting test pig screening and big difference between test results in the traditional test for the efficacy of the porcine circovirus 2-type vaccine. The test method provided by the invention has the advantages of short test time, simple operation, low cost, accurate result, high repeatability, high application value and wide application prospect.

Description

A kind of porcine circovirus type 2 vaccines efficacy test method
The application is the divisional application of patent application 201010210104.7, and the original bill applying date is on June 22nd, 2010, former Case entitled " a kind of porcine circovirus type 2 vaccines efficacy test method ".
Technical field
The present invention relates to biological technical field, particularly a kind of porcine circovirus type 2 vaccines efficacy test method.
Background technology
Porcine circovirus 2 type (porcine cirovirus type 2, PCV2) is to cause weaned piglet multisystem exhaustion The main pathogen of syndrome (post-weaning multisystemic wasting syndrome, PMWS).From 1991 Since breaking out in Canadian swinery, PMWS has involved the whole world, and to whole world pig industry huge economic loss is caused.PCV2 It is also scorching related with nephrotic syndrome and PRDC to Hypertrophic necrotizing pneumonia, sow breeding difficulty, pigskin. Porcine circovirus associated diseases (PCV2Associated Disease, PCVD), mainly PMWS was caused in 2005 to Europe Loss be up to 600,000,000 Euros.PCV2 infectious diseases causes the death rate to increase, the price of deed is reduced, and adds China scale pig Field feeding and management level comparatively falls behind, therefore PCVD also causes huge economic loss to the pig industry of China.
At present, the PCV2 vaccines for listing in the world are divided into inactivated vaccine, subunit vaccine and chimeric inactivated vaccine.PCV2 vaccines The method that efficacy test method is typically with piglet challenge viral dosage.And China swinery PCV2 is seriously polluted, for filtering out conjunction The experiment pig for PCV2 vaccine developments or vaccine potency inspection of lattice is relatively difficult, and piglet challenge viral dosage method is present Time-consuming, laborious, expensive and PCV2 attacks malicious model and sets up the problems such as difficult, assay differences between batches are big.
The content of the invention
In consideration of it, the present invention is by porcine circovirus type 2 vaccines immunity small white mouse, PCV2 specificity in its serum is determined anti- A kind of body level, there is provided simple and direct effective PCV2 vaccine potency test methods.Comprise the steps：
1. the immunity of small white mouse
Porcine circovirus type 2 vaccines are percutaneously descended immunity small white mouse, according still further to identical immunization route and agent after 10-21 days Amount carries out booster immunization once；
The measure of 2 small white mouse antibody horizontals
After booster immunization 14-28 days, small white mouse is put to death, gather and separate serum, determine antibody horizontal；
In the method for inspection of porcine circovirus 2 type inactivated vaccine effect of the present invention, the immunizing agent of the small white mouse Measure as 0.1-0.2ml, and points 4 points immune.
In porcine circovirus type 2 vaccines efficacy test method of the present invention, the measure of the small white mouse antibody horizontal Method includes following operating procedure,
1. serum to be checked is done into doubling dilution with PBS；
2. in adding the ELISA ELISA Plates of coated Porcine circovirus type 2 Cap；
3. 37 DEG C of incubators are put into, 1h is acted on；
4. discard one to resist, PBST washings；
5. the HRP mark sheep anti-mouse iggs (two resist) of PBS dilutions are added, 37 DEG C of incubators are put into, 1h is acted on；
6. two are discarded to resist, is washed with PBST；
7. nitrite ion is added to be developed the color；
8. terminate liquid color development stopping is added；
9. ELIASA reads OD450, judge vaccine potency；
The dilution, cleaning solution, nitrite ion, terminate liquid are the routine liquid of prior art.
Optimally, the time of first immunisation small white mouse of the present invention is 14 days.
Optimally, the time of booster immunization small white mouse of the present invention is 21 days.
The present invention has the following technical effect that：
1. detection time is short, and than the time of efficacy test in pig body 20 days are at least reduced；
2. the inventive method is simple to operate, favorable repeatability, as a result accurately.
3. small white mouse inheritance stability, the experimental result significant difference for being not in animal used as test individual difference and causing；
4. simple to operate with vaccine immunity small white mouse, without the need for substantial amounts of experiment pig is bought, greatly save cost, people Power, improves the efficiency of vaccine detection.
Specific embodiment
Immune small white mouse antibody horizontal, immune swine antibody are established in embodiments of the invention using the method for parallel laboratory test Level and piglet attack the relation of poison protection, find after immune small white mouse antibody horizontal and immunity the antibody horizontal of pig and attack after poison to drench Fawn on infection and there is parallel relation.Using the method for the present invention, the PCV2 vaccine immunity pig antibody water of usual employing can be substituted It is plain to test and piglet attacks malicious Protection.Also PCV2 vaccines effect is made in the embodiment of the present invention using detection method The criterion of acceptability of power, is that the large-scale production and detection of PCV2 vaccines provides the foundation.
Embodiment 1
The detection of porcine circovirus 2 type inactivated vaccine effect
1. inactivated vaccine is originated
The strain of PCV2 inactivated vaccines is PCV2-SH strains (preserving number CGMCC No.2389).By PCV2-SH strains inoculation PK15 cells, harvest viral antigen liquid as inactivated vaccine after 5 days.
By the inactivated vaccine of preparation be divided into three group I, II, III, its antigenic content Jing determine be respectively 105.0TCID50/ml、 105.5TCID50/ml、106.0TCID50/ml.Below with three groups of inactivated vaccines as sample, immune small white mouse determines its serum antibody Level.
2. immune small white mouse antibody test
(1) immunity of BALB/c small white mouses
The BALB/c mouse of 5-6 week old health is randomly divided into into 4 groups, 5 per group, I group adopts antigenic content 105.0TCID50The porcine circovirus 2 type inactivated vaccine immunity of/ml, II group adopts antigenic content 105.5TCID50The pig annulus of/ml Viral 2 type inactivated vaccines immunity, III group adopts antigenic content 106.0TCID50The porcine circovirus 2 type inactivated vaccine immunity of/ml, IV group is blank group.Divide 4 points of immune, each point immunity 0.05ml, strengthen according still further to identical immunization route and dosage after 14 days Immunity is once.
(2) measure of small white mouse antibody horizontal
After booster immunization 21 days, by extracing eyeball, collection separates serum, determines its antibody horizontal.Operating procedure is as follows：
1. serum dilution to be checked does 1:50 times of dilutions, then do again 2 times and are serially diluted, to 1:102400；
2. the serum after dilution is separately added in the ELISA ELISA Plates of coated Porcine circovirus type 2 Cap, PCV2Cap eggs Obtained by escherichia expression system expression and purification in vain, package amount is 0.5 μ g/ holes, step 1. in 12 dilution factors each dilutions Degree plus 1 hole, per the μ l of hole 100.Set up the identical dilution negative serum control group of small white mouse simultaneously；
3. 37 DEG C of incubators are put into, 1h is acted on；
4. discard one to resist, add cleaning solution, 300 μ l/ holes are washed 3 times, each 3-5min；
5. addition dilution makees horseradish peroxidase (HRP) the mark sheep anti-mouse igg (two resist) of 5000 times of dilutions, 100 μ l/ holes；37 DEG C of incubators are put into, 1h is acted on；
6. two are discarded to resist, with cleaning solution, is washed 3 times, each 3-5min；
7. the μ l/ holes of nitrite ion 100 are added, room temperature lucifuge colour developing 10-15min is carried out；
8. the μ l/ holes of terminate liquid 100, color development stopping are added；
9. ELIASA reads OD450；
10. result judgement method：Serum OD to be checked450/ with dilution factor negative serum OD450>=2.1 are judged to positive findings； Serum OD to be checked450/ with dilution factor negative serum OD450≤ 2.1 are judged to negative findings.Done with the greatest dilution of positive findings For the antibody horizontal of small white mouse.
Small white mouse antibody horizontal testing result is as follows：
I groups：The antibody titer of 5 small white mouses is respectively 1:200、1:200、1:200、1:200、1:400；II group 5 little The antibody titer of white mouse is respectively 1:400、1:400、1:400、1:800、1:800；The antibody titer difference of III group of 5 small white mouse For 1:800、1:800、1:800、1:800、1:1600；IV group be the antibody titer of 5 small white mouses of blank group≤1:50.
Solution used is as follows：
Dilution formula of liquid：
2.9 grams of disodium hydrogen phosphate
0.2 gram of potassium dihydrogen phosphate
8.0 grams of sodium chloride
0.2 gram of potassium chloride
Add water and be settled to 1000ml.
Washing formula of liquid：
2.9 grams of disodium hydrogen phosphate
0.2 gram of potassium dihydrogen phosphate
8.0 grams of sodium chloride
0.2 gram of potassium chloride
Tween-20 0.5ml
Add water and be settled to 1000ml.
Colour developing formula of liquid：
Tmb substrate liquid：(1mg/ml)
TMB powder 0.01g
DMSO 10ml
Tmb substrate colorbuffer (pH5.0,100ml)
Citric acid：1.02 gram
3.69 grams of disodium hydrogen phosphate
Nitrite ion：Lucifuge develops the color
Tmb substrate liquid 1ml
Tmb substrate colorbuffer 9ml
The μ l of 30% hydrogen peroxide 10
Terminate formula of liquid：2mol/L sulfuric acid
3. porcine circovirus 2 type inactivated vaccine immune swine antibody horizontal is determined
(1) immunity of experiment pig
By the PCV2 antigen-antibodies feminine gender of 21 ages in days health and pig blue-ear disease antigen-antibody feminine gender weanling pig, it is randomly divided into 4 groups, 5 per group, I group adopts antigenic content 105.0TCID50The porcine circovirus 2 type inactivated vaccine immunity of/ml, II group using anti- Former content 105.5TCID50The porcine circovirus 2 type inactivated vaccine immunity of/ml, III group adopts antigenic content 106.0TCID50/ ml's Porcine circovirus 2 type inactivated vaccine immunity, IV group is blank group.Per immunity 1ml, according still further to identical immunization route after 14 days With dosage booster immunization once.
(2) measure of pig antibody horizontal
After booster immunization 21 days, taken a blood sample by vena cava anterior, separated serum, determined its antibody horizontal.TPPA Method operating procedure is as follows：
1. serum dilution to be checked does 1:50 times of dilutions, then do again 2 times and are serially diluted, to 1：102400；
2. the serum after dilution is separately added in the ELISA ELISA Plates of coated Porcine circovirus type 2 Cap, PCV2Cap eggs Obtained by escherichia expression system expression and purification in vain, package amount is 0.5 μ g/96 holes, each dilution factor adds 1 hole, 100 μ l/ holes, Simultaneously piglet negative serum control is set up, do same dilution factor；
3. 37 DEG C of incubators are put into, 1h is acted on；
4. discard one to resist, add cleaning solution, 300 μ l/ holes are washed 3 times, each 3-5min；
5. addition dilution does horseradish peroxidase (HRP) the mark rabbit-anti pig IgG (two resist) of 10000 times of dilutions, 100 μ l/ holes；37 DEG C of incubators are put into, 1h is acted on；
6. two are discarded to resist, cleaning solution is used；Washing 3 times, each 3-5min；
7. the μ l/ holes of TMB nitrite ions 100 are added, room temperature lucifuge colour developing 10-15min is carried out；
8. 2mol/L H are added2SO4The μ l/ holes of solution 100, color development stopping；
9. ELIASA reads OD450；
10. result judgement method：Serum OD to be checked450/ with dilution factor negative serum OD450>=2.1 are judged to positive findings； Serum OD to be checked450/ with dilution factor negative serum OD450≤ 2.1 are judged to negative findings.Done with the greatest dilution of positive findings For the antibody horizontal of small white mouse.
Pig antibody horizontal testing result is as follows：
I groups：The antibody titer of 5 pigs is respectively 1:1600、1:1600、1:800、1:3200、1:6400；II group of 5 pig Antibody titer be respectively 1:3200、1:3200、1:3200、1:3200、1:6400；The antibody titer of III group of 5 pig is respectively 1:3200、1:3200、1:6400、1:6400、1:12800；IV group is that the antibody titer of 5 pigs of blank group is≤1:50.
4. piglet protest test
At present, PCV2 attacks malicious model and is difficult to set up, and is because that its clinical symptoms are not obvious, is clinically primarily due to PCV2 and other cause of disease mixed infections, so it is difficult to checking the infection state of PCV2 by clinical symptoms.And PCV2 is in pig body Main target tissue is lymphoid tissue.Therefore, the present embodiment is attacked the hilar lymph node of piglet or groin after poison and is drenched using collection Fawn on, immunohistochemistry (IHC) method detects its antigen status, judge that piglet attacks poison protection result with this.
All pigs in immune group and blank group are carried out into PCV2-SH strains (106.0TCID50/ ml) attack poison.Every pig drop Nose 1ml, musculi colli injection 2ml, isolated rearing.Attack after poison the 4th, 7 days it is not complete in two oxters and two buttock injections Freunds respectively The keyhole hemocyanin (4mg/ml, 0.5ml) and lumbar injection thioglycollate medium 10ml of full adjuvant emulsion.At the 11st day With 19 days lumbar injection thioglycollate medium 10ml.Attacking 25 days after poison carries out cuing open killing, and gathers hilar lymph node or groin Lymph node is fixed, and then carries out antigen detection with IHC methods, compares infection PCV2 in blank group and immune group lymph node and resists Former situation, judges that piglet attacks poison protection result with this.Result of the test is shown in Table 1.
By 3 batches of porcine circovirus 2 type inactivated vaccine immunity small white mouse antibody horizontals and immune swine antibody horizontal and Piglet attacks the result of malicious Protection, compares, and the results are shown in Table 1.
The small white mouse antibody horizontal of table 1. attacks the relation of malicious Protection with pig antibody horizontal and piglet
By 3 groups of porcine circovirus type 2 vaccines immunity BALB/c mouse and PCV2 antigen-antibodies by different antigenic contents Double-negative weanling pig, by relative immunity small white mouse antibody horizontal and immune swine antibody horizontal, finds what both produced PCV2 antibody has parallel relation, i.e., immune small white mouse antibody horizontal is presented positively related relation with immune swine antibody horizontal.
Protest test is carried out to the piglet after immunity, by the detection to PCV2 antigens in lymph node, has been found young Pig is attacked the ratio shared by the cell of the PCV2 infected in lymph node after poison and has parallel relation with immune small white mouse antibody horizontal, i.e., Immune small white mouse antibody horizontal is higher, and the ratio shared by the cell of the PCV2 infected in lymph node is less, illustrates to show protectiveness It is good；Adverse immune small white mouse antibody horizontal is lower, and the ratio shared by the cell of the PCV2 infected in lymph node is bigger, and explanation shows Protectiveness is poor.
The piglet immunological protest test result of the vaccine of 3 batches of different antigenic contents shows that antigenic content is 105.0TCID50The vaccine of/ml can provide the protection of piglet 40%, and antigenic content is 105.5TCID50The vaccine of/ml can be with 80% protection is provided, antigenic content is 106.0The vaccine of TCID50/ml can provide 100% protection.We are not yet The result and small white mouse antibody horizontal for being difficult to see protest test has parallel relation.Therefore deduce that simultaneously 105.5TCID50The antigen of/ml is Minimal Protective dosage, and the vaccine immunity small white mouse antibody horizontal under this dosage is 1:400 or 1:800.Thus, the criterion of acceptability of PCV2 vaccine potencies can be made using detection method.In the present embodiment, adopt Method of the present invention detection small white mouse antibody horizontal is more than 1:400, as the qualified standard of sample vaccine test, you can to reach 80% protective rate, the effectively purpose of defence PCVD are produced to swinery.
In sum, it can be deduced that the antibody horizontal of immune small white mouse antibody horizontal and pig and attack Lymphod infection after poison and deposit In parallel relation.Using PCV2 vaccine immunity small white mouses, PCV2 specific antibody levels experiment in its serum is determined, can be substituted The experiment of vaccine immunity pig antibody horizontal and piglet that existing porcine circovirus 2 type inactivated vaccine efficacy test method is generally adopted Attack malicious Protection.
The method of inspection of the vaccine potency of the present invention, first immunisation is not limited to 14 days with the time interval of booster immunization, Booster immunization, the knot of the inspection of its vaccine potency are carried out after first immunisation 10-21 days according still further to identical immunization route and dosage Result no significant difference of the fruit with 14 days.It is not limited to 21 days with the time interval for putting to death mouse collection blood sample after booster immunization, After booster immunization 14-28 days, small white mouse is put to death, gather and separate serum, knot of the result of the inspection of its vaccine potency with 21 days Fruit no significant difference.
The method of inspection of vaccine potency of the present invention, the immunizing dose of small white mouse is not limited to 0.05ml, immunizing agent Measure for 0.1-0.2ml when, the result of the inspection of vaccine potency and immunizing dose for 0.05ml result no significant difference.
The method of the detection vaccine of the present invention, is not limited to be applied to PCV2 inactivated vaccines, because other kinds of PCV2 epidemic diseases Seedling such as recombinant vaccine, subunit vaccine can also cause immune response in Mice, other types PCV2 vaccine, such as Recombinant vaccine, subunit vaccine can be detected with the method for the present invention.
Porcine circovirus 2 type inactivated vaccine immunity small white mouse antibody horizontal assay method detection time is short, simple to operate, into This is low, and the result for obtaining is accurate, favorable repeatability, with good using value and wide market prospects.

Claims (5)

1. a kind of porcine circovirus 2 type inactivated vaccine efficacy test method, it is characterised in that step is as follows：
(1) small white mouse antibody horizontal is determined
1. the immunity of small white mouse
Porcine circovirus type 2 vaccines are percutaneously descended immunity small white mouse, is entered according still further to identical immunization route and dosage after 10-21 days Row booster immunization is once；
2. the measure of small white mouse antibody horizontal
Booster immunization, after 14-28 days, puts to death small white mouse, gathers and separate serum, determines antibody horizontal；
(2) judge that vaccine potency checks whether qualified method by determining the antibody horizontal of small white mouse
1. immune small white mouse antibody horizontal and immune swine antibody horizontal, have parallel relation between the result of protest test；
2. judge that the vaccine potency is checked whether according to the small white mouse antibody horizontal for determining qualified, the detection small white mouse antibody water It is flat to be more than 1:400, as the qualified standard of sample vaccine test, you can produce 80% protective rate to swinery to reach.
2. porcine circovirus 2 type inactivated vaccine efficacy test method according to claim 1, it is characterised in that step (1) The Best Times of middle first immunisation are 14 days.
3. porcine circovirus 2 type inactivated vaccine efficacy test method according to claim 1, it is characterised in that step (1) The Best Times of middle booster immunization are 21 days.
4. porcine circovirus 2 type inactivated vaccine efficacy test method according to claim 1, it is characterised in that step (1) Described in the immunizing dose of vaccine be 0.1-0.2ml, and points 4 points immune.
5. porcine circovirus 2 type inactivated vaccine efficacy test method according to claim 1, it is characterised in that described anti- The assay method of body level includes following operating procedure,
1. serum to be checked is done into doubling dilution with PBS；
2. in adding the ELISA ELISA Plates of coated Porcine circovirus type 2 Cap；
3. 37 DEG C of incubators are put into, 1h is acted on；
4. discard one to resist, PBST washings；
5. the HRP mark sheep anti-mouse iggs (two resist) of PBS dilutions are added, 37 DEG C of incubators are put into, 1h is acted on；
6. two are discarded to resist, is washed with PBST；
7. nitrite ion is added to be developed the color；
8. terminate liquid color development stopping is added；
9. ELIASA reads OD450, judge vaccine potency.
CN201611140272.7A 2010-01-28 2010-06-22 Method for testing efficacy of porcine circovirus 2-type vaccine Pending CN106645716A (en)

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