CN103336116B - A kind of method for testing efficacy of swine fever live vaccine - Google Patents
A kind of method for testing efficacy of swine fever live vaccine Download PDFInfo
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- CN103336116B CN103336116B CN201310239545.3A CN201310239545A CN103336116B CN 103336116 B CN103336116 B CN 103336116B CN 201310239545 A CN201310239545 A CN 201310239545A CN 103336116 B CN103336116 B CN 103336116B
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Abstract
The invention belongs to technical field of new biological veterinary medicaments, be specifically related to the method for testing efficacy of live vaccines of hog cholera.The effect of live vaccine is evaluated by the viral level detecting live vaccine by indirect immunofluorescence (IFA) method.The method is short for detection time, and simple to operate, cost is low, the result that obtains accurate, favorable repeatability, has good using value and wide market outlook.
Description
Technical field
The present invention relates to biological technical field, particularly a kind of method for testing efficacy of swine fever live vaccine.
Background technology
Swine fever (ClassicalSwinefever, be called for short CSF) be also called hog cholera (Hogcholera) or Europe class swine fever (Europeanswinefever), it is the viral infectious disease of the acute or super febris acuta of a boar, feature clinically disseminates rapid, fever, can see typical hemorrhage pathology when dissecting inspection.Swine fever can infect the pig at various age, popular throughout the year, and M & M is all high, very harmful to pig.This disease threatens one of most important infectious disease of pig industry.Swine fever is classified as one of category-A 16 kinds of Notifiable diseases by OIE (OIE), and China is then decided to be a class deadly infectious disease.Many countries and regions use live vaccines of hog cholera to force immunoprophylaxis, are also the best approaches of preventing and treating swine fever at present.
At present, China produces live vaccines of hog cholera effect detection method is all adopt rabbit precursor reactant by the use of thermal means.Not only need much to test White Rabbit, time-consumingly to require great effort again, and due to the minimizing gradually of China's pure lines large ear rabbit and strain more and more assorted, cause nowadays being difficult to by the hot effect accurately detecting live vaccine of rabbit precursor reactant.
Summary of the invention
Given this, the invention provides a kind of new method for testing efficacy of swine fever live vaccine, specifically, by detecting by indirect immunofluorescence (IFA) method the effect that live vaccine viral level in passage cell evaluates live vaccine, substitute traditional rabbit precursor reactant by the use of thermal means, to reach quick and precisely, the simple and effective object detecting swine fever vaccine potency while still alive.
The invention provides a kind of method for testing efficacy of swine fever live vaccine, comprise the steps:
(1) passage cell is prepared
To the cell of individual layer be covered with, with EDTA-trypsinization dispersion, obtain passage cell suspension;
(2) viral level of live vaccines of hog cholera in passage cell measures
With cell maintenance medium, freeze-drying live vaccines of hog cholera back dissolving is diluted, cell suspension in live vaccine dilution and step (1) is added in Tissue Culture Plate, is placed in incubator and cultivates, set up simultaneously and do not connect malicious normal cell controls;
(3) viral level in indirect immunofluorescence (IFA) method detecting step (2) gained cell
1. Tissue Culture Plate is taken out from incubator, discard and cultivate liquid in plate hole, wash with PBS;
2. add aqueous acetone solution to fix;
3. discard aqueous acetone solution, wash with PBS;
4. the swine fever positive serum (primary antibodie) of PBS dilution is added;
5. discard primary antibodie, PBS washs;
6. the FITC adding PBS dilution marks the anti-pig IgG of rabbit (two resist);
7. discard two to resist, with PBS washing, finally add PBS and preserve;
8. be placed in fluorescence microscopy Microscopic observation fluorescing matter, judge live vaccines of hog cholera effect.
Preferably, while adding cell suspension in step (1) in cell plates, add diluted live vaccines of hog cholera liquid in step (2).
Preferably, the dilution of live vaccines of hog cholera described in step (2) carries out under 0-8 DEG C of low temperature environment.
Preferably, in step (2), incubator is CO
2incubator, temperature is 36-37 DEG C, CO
2content is 2.5%-5%, and incubation time is 2-5 days.
Preferably, in step (3), the number of times of PBS washing is 2-7 time.
Preferably, in step (3), aqueous acetone solution temperature is 0-8 DEG C, and concentration is 30%-90%.
Preferably, in step (3), the extension rate range of choice of primary antibodie is 50-1000 times, and the two extension rate ranges of choice resisted are 50-10000 times.
Preferably, in step (3), the extension rate of primary antibodie is 400 times, and two extension rates resisted are 500 times.
Compared with prior art, the present invention has following technique effect:
1. detection time is short, only needs 3 days, and usually at least needs 11 days with the detection of rabbit body infective dose.
2., by cell infection virus, simple to operate, cost is low, does not need to buy a large amount of animals used as test, does not need special messenger's determination experiment animal heat simultaneously.
3. utilize cell infection virus, condition be homogeneous, stable, there will not be animal used as test individual difference and the experimental result significant difference that causes.
Embodiment
Embodiment 1
Method for testing efficacy of swine fever live vaccine
1. prepare passage cell
Pig testis (ST) cell covering with individual layer (is derived from ATCC, U.S. typical case bacterium kind preservation center), α-MEM the cell maintenance culture solution containing 3% hyclone is added after pancreatin (EDTA containing 0.02%) digestion dispersion with 0.25%, after cell count, cell suspension is joined in 96 porocyte plates, every hole 100 μ l.
2. the dilution of live vaccines of hog cholera
The present embodiment has prepared three mass products vaccines, and determines its antigenic content respectively.
The strain of live vaccines of hog cholera is fever virus lapinized Chinese Strain (China Veterinery Drug Inspection Office's preservation, preserving number AV1412).Fever virus lapinized Chinese Strain is inoculated ST cell, the viral antigen liquid of results is equipped with suitable freeze drying protectant and makes live vaccines of hog cholera.With containing the α-MEM cell maintenance culture solution of 3% hyclone by freeze-drying live vaccines of hog cholera back dissolving, on ice or other can keep in the low temperature environment of 0-8 DEG C, carrying out 10 times of serial dilutions, get 10
-1-10
-7dilutability adds in 96 orifice plates of the Tissue Culture Plate in step 1, every hole 100 μ l, and each dilutability does 8 holes and repeats, and Tissue Culture Plate is placed in CO
2cultivate in incubator, temperature is 36-37 DEG C, CO
2content is 2.5%-5%, and incubation time is 2-5 days.Preferably, temperature is 37 DEG C, CO
2content is 5%, incubation time is 3 days, can carry out indirect immunofluorescence (IFA) and judge malicious valency.Meanwhile, set up and do not connect malicious normal cell controls.Concrete steps are as follows:
(1) discarding liquid in 96 orifice plates, is 7.4 by the PBS(pH value of 0.01mol/L) wash 2-7 time, each 3min, optimally, washing times is 3 times, each 3min;
(2) the 30%-90% aqueous acetone solution that temperature is 0-8 DEG C is added, optimally, the aqueous acetone solution 100 μ l/ hole of 80%, 30min fixed by 4 DEG C of refrigerators;
(3) discarding acetone, is 7.4 by the PBS(pH value of 0.01mol/L) wash 2-7 time, each 3min, optimally, washing times is 3 times, each 3min;
(4) adding by the PBS(pH value of 0.01mol/L is 7.4) make the swine fever positive serum (primary antibodie, purchased from China Veterinery Drug Inspection Office) that 50-1000 doubly dilutes, optimally, extension rate is 400 times, antibody content 5 μ g/ml, every hole 100 μ l, 37 DEG C of effect 60min;
(5) discarding primary antibodie, is 7.4 by the PBS(pH value of 0.01mol/L) wash 2-7 time, each 3min, optimally, washing times is 3 times, each 3min;
(6) adding by the PBS(pH value of 0.01mol/L is 7.4) make fluorescein isothiocynate (FITC) that 50-10000 doubly dilutes and mark the anti-pig IgG of rabbit (two resist, available from Sigma), optimally, extension rate is 500 times, antibody content 6 μ g/ml, every hole 100 μ l, 37 DEG C of effect 60min;
(7) discarding two to resist, is 7.4 by the PBS(pH value of 0.01mol/L) wash 3 times, each 10min, the PBS(pH value finally adding 0.01mol/L is 7.4), every hole 50 μ l;
(8) be placed in fluorescence microscopy Microscopic observation fluorescing matter, record fluorescence hole count, calculates TCID according to Reed-Muench method
50result;
(9) IFA fluorescence criterion: special yellow-green fluorescence does not appear in not connecing in malicious normal control cells endochylema of setting up, and connect poison cell plate hole inner cell endochylema in can be observed specific yellow-green fluorescence, be judged to be positive hole.Observations is as following table 1.
Table 1 indirect immunofluorescence (IFA) observations
Calculate TCID50 result according to Reed-Muench method to the following is shown in table 2.
The malicious valency measurement result of table 2 three mass products vaccine
| Batches | (every part is containing TCID for poison valency measurement result 50） |
| I batch | 10 4.5TCID 50/ head part |
| II batch | 10 4.667TCID 50/ head part |
| III batch | 10 4.57TCID 50/ head part |
3. animal challenge viral dosage
(1) live vaccine dilution: concrete dilution process following (every bottle of vaccine calculates by 20 parts):
I, II, III batch of vaccine, two bottles of freeze dried vaccine physiological saline mix back dissolving to 40ml, every part 1ml, the vaccine liquid got after wherein 1ml back dissolving adds 5.325ml, 8.290ml, 6.431ml physiological saline respectively, and then carry out 10 times of serial dilutions and 5 times of dilutions successively, the most often criticize vaccine dilution for viral level 5TCID
50/ ml, 1TCID
50/ ml, 0.2TCID
50the vaccine of/ml uses liquid.Concrete operations are as follows:
After getting dilution, vaccine liquid 1ml adds 9ml physiological saline; After getting step dilution, vaccine liquid 1ml adds 9ml physiological saline; After getting step dilution, vaccine liquid 1ml adds 9ml physiological saline; After getting step dilution, vaccine liquid 2ml adds 8ml physiological saline; After getting step dilution, vaccine liquid 2ml adds 8ml physiological saline.
(2) animal experiment design
This tests with 39 piglets (swine fever antigen antibody is feminine gender, body weight is even, healthy), and be divided into 10 groups at random, attacking malicious control group is 3, and immune group often organizes 4.Often criticize vaccine immune 3 groups respectively, immunizing dose is respectively 0.2TCID
50/ head part, 1TCID
50/ head part, 5TCID
50/ head part.Latter 14 days of immunity, immune group injects 10 together with the control group that condition is identical
5the swine fever crossdrift system blood poison 1ml (10 of minimum lethal dose (MLD)
5mLD refers to swine fever crossdrift system blood poison dilution 10
5attack malicious 1ml to pig doubly, pig is still dead, is to attack malicious standard in industry).Observe 16, result of determination, as shown in table 3 below.According to result, add up malicious valency and attack the correlativity between malicious protection, count minimum immune dosage.
The design of table 3 animal experiment and result
The result of challenge viral dosage shows, I, II, III batch of live vaccines of hog cholera (ST passage cell) is diluted to 1TCID respectively
50/ ml, injection 1ml, can reach the total protective number of piglet.Namely the minimum immune dosage of 3 mass products vaccines to the strong poison of swine fever crossdrift system is 1TCID
50/ head part.Current hog cholera vaccine national standard is all that after diluting 3000 times with the vaccine an of part, immune swine still can resist strong virus attack, and according to the inventive method, tires be more than or equal to 10 when the live vaccines of hog cholera detected
3.5tCID
50during/head part, immune swine after diluting 3000 times, can reach total protected effect, meets the examination criteria of country.Therefore, process according to the invention, can propose to identify that live vaccines of hog cholera meets the standard of country's detection.
In sum, the invention provides a kind of new method for testing efficacy of swine fever live vaccine, specifically, by detecting by indirect immunofluorescence (IFA) method the effect that live vaccine viral level in passage cell evaluates live vaccine.The method is short for detection time, and simple to operate, cost is low, the result that obtains accurate, favorable repeatability, has good using value and wide market outlook.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (7)
1. a method for testing efficacy of swine fever live vaccine, is characterized in that: comprise the steps:
(1) passage cell is prepared
To the cell of individual layer be covered with, with EDTA-trypsinization dispersion, obtain passage cell suspension;
(2) viral level of live vaccines of hog cholera in passage cell measures
With cell maintenance medium, freeze-drying live vaccines of hog cholera back dissolving is diluted, cell suspension in live vaccine dilution and step (1) is added in Tissue Culture Plate, be placed in incubator to cultivate, set up simultaneously and do not connect malicious normal cell controls, wherein, described live vaccines of hog cholera is fever virus lapinized Chinese Strain AV1412;
(3) viral level in indirect immunofluorescence (IFA) method detecting step (2) gained cell
1. Tissue Culture Plate is taken out from incubator, discard and cultivate liquid in plate hole, wash with PBS;
2. add aqueous acetone solution to fix;
3. discard aqueous acetone solution, wash with PBS;
4. the swine fever positive serum of PBS dilution is added, i.e. primary antibodie;
5. discard primary antibodie, PBS washs;
6. the FITC adding PBS dilution marks the anti-pig IgG of rabbit, and namely two resist;
7. discard two to resist, with PBS washing, finally add PBS and preserve;
8. be placed in fluorescence microscopy Microscopic observation fluorescing matter, judge live vaccines of hog cholera effect.
2. method for testing efficacy of swine fever live vaccine according to claim 1, is characterized in that: while adding cell suspension in step (2) in Tissue Culture Plate, adds diluted hog cholera vaccine liquid in step (2).
3. method for testing efficacy of swine fever live vaccine according to claim 1, is characterized in that: the dilution of live vaccines of hog cholera described in step (2) is carried out under 0-8 DEG C of low temperature environment.
4. method for testing efficacy of swine fever live vaccine according to claim 1, is characterized in that: in step (2), incubator is CO
2incubator, temperature is 36-37 DEG C, CO
2content is 2.5%-5%, and incubation time is 2-5 days.
5. method for testing efficacy of swine fever live vaccine according to claim 1, is characterized in that: in step (3), the number of times of PBS washing is 2-7 time.
6. method for testing efficacy of swine fever live vaccine according to claim 1, is characterized in that: in step (3), aqueous acetone solution temperature is 0-8 DEG C, and concentration is 30%-90%.
7. method for testing efficacy of swine fever live vaccine according to claim 1, is characterized in that: in step (3), the extension rate range of choice of primary antibodie is 50-1000 times, and the two extension rate ranges of choice resisted are 50-10000 times.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102600469A (en) * | 2012-03-07 | 2012-07-25 | 齐鲁动物保健品有限公司 | Classical swine fever live vaccine |
| CN103908665B (en) * | 2013-01-05 | 2016-05-25 | 普莱柯生物工程股份有限公司 | A kind of vaccine combination and its preparation method and application |
| CN103105493A (en) * | 2013-02-07 | 2013-05-15 | 新疆天康畜牧生物技术股份有限公司 | Novel method for replacing detection of rabbit thermal response of hog cholera lapinized virus strain |
| CN103616505B (en) * | 2013-12-10 | 2016-01-27 | 瑞普（保定）生物药业有限公司 | A kind of hog cholera lapinised virus live vaccine effect detection method |
| CN103698518A (en) * | 2013-12-30 | 2014-04-02 | 山东滨州博莱威生物技术有限公司 | Method for detecting virus content of swine fever live vaccine through indirect immunofluorescence |
| CN104535764B (en) * | 2014-12-07 | 2016-05-18 | 青岛易邦生物工程有限公司 | A kind of CSFV gene recombinant adenovirus vector vaccine virus content assaying method |
| CN104407136B (en) * | 2014-12-07 | 2016-05-18 | 青岛易邦生物工程有限公司 | Viral level assay method in a kind of encephalomyelitis of chicken live vaccine |
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| CN101846683B (en) | 2014-08-06 |
| CN103336116A (en) | 2013-10-02 |
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