CN104689311A - Method for producing avian encephalomyelitis virus inactivated vaccine - Google Patents
Method for producing avian encephalomyelitis virus inactivated vaccine Download PDFInfo
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- CN104689311A CN104689311A CN201410846776.5A CN201410846776A CN104689311A CN 104689311 A CN104689311 A CN 104689311A CN 201410846776 A CN201410846776 A CN 201410846776A CN 104689311 A CN104689311 A CN 104689311A
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Abstract
The invention discloses a method for producing an avian encephalomyelitis virus inactivated vaccine. The method comprises the following steps: a. selecting cell lines to serve as cells for preparing the vaccine; b. passing and culturing the cells for preparing the vaccine; c. breeding a virus seed for production; d. producing venom for preparing the vaccine; and e. preparing the vaccine. According to the method disclosed by the invention, continuous cell lines are used as the cells for preparing the vaccine, and sensitive cell lines are screened to reinforce the match of the virus and the cells. The method disclosed by the invention has the advantages of simple and stable production process, easiness in operation, small batch difference and low cost, and the safety and immunogenicity of the produced avian encephalomyelitis virus inactivated vaccine are good.
Description
Technical field
The present invention relates to a kind of method that cell line produces avian encephalomyclitis virus inactivated vaccine, belong to biological technical field.
Background technology
Avian encephalomyelitis (Avian Encephalomyelitis; AE) also known as epidemic tremor, it is chickling below 4 week age of a kind of main harm of being caused by avian encephalomyclitis virus (AEV), to encroach on chickling central nervous system, cause chickling apyetous encephalitis to be the viral infectious of major pathologic features, sick young main manifestations is movement disorder, head and neck trembles and the nervous symptoms such as hallux paralysis.There is features such as occurring suddenly and be difficult to the prediction duration, the loss of chickling and the decline of hen laying rate can be caused, become one of main epidemic disease our times endangering aviculture development.China since there were this pathogenetic report 80 mid-nineties 90s, all have throughout the country occur and popular, cause larger loss to poultry husbandry.This disease there is no effective medicine at present, and therefore epidemic prevention seems even more important.
Avian encephalomyclitis virus (AEV) belongs to Picornaviridae, hepatovirus, can high-titer breeding in the chickling of susceptible chicken group, Embryo Gallus domesticus.The research report of avian encephalomyclitis virus is cultivated in the primary Embryo Gallus domesticus source cell of existing application (chick embryo fibroblast, nephrocyte, neurogliocyte, pancreatic cell etc.) both at home and abroad, but virus titer is low, is all no more than 10
3.5eID
50/ ml.Also do not have successful Application cell line to cultivate the report of avian encephalomyclitis virus, current avian encephalomyclitis virus vaccine preparation all adopts chick embryo method.Use chick embryo culture avian encephalomyclitis virus, need with a large amount of SPF Embryo Gallus domesticus, production cost is high, labor intensity is large, product differences between batches are large, bring difficulty to the raising of vaccine Yield and quality.
Summary of the invention
Technical problem to be solved by this invention is that the defect overcoming prior art provides a kind of method utilizing cell line to produce fowl marrowbrain viral inactivation vaccine.
Technical problem of the present invention is solved by following technical scheme.
Produce a method for avian encephalomyclitis virus inactivated vaccine, comprise the steps:
A. select cell line as seedling cell;
B. the going down to posterity and cultivation of seedling cell
When seedling cell forms good monolayer, after EDTA-trypsinization, adding cell growth medium and be prepared into cell suspension, going down to posterity or virus inoculation for continuing;
C. the breeding of production seed culture of viruses
By seedling cell suspension, subpackage Tissue Culture Flask, Simultaneous vaccination avian encephalomyelitis chicken embryo tissue poison, changes cell maintenance medium when cultivating 72h, continues cultivation 48 ~ 72h, and results culture is generation cell toxicant; Generation cell toxicant Secondary Culture is secondary cell toxicant; Get a generation, secondary cell toxicant as production seed culture of viruses;
D. production seedling venom
By seedling cell suspension, subpackage Tissue Culture Flask, accesses avian encephalomyclitis virus production seed culture of viruses simultaneously and cultivates, change cell maintenance medium, continue cultivation 48 ~ 72h during 72h, results culture ,-15 DEG C of preservations.
E. vaccine preparation
Add adjuvant by after the seedling venom deactivation of results, emulsifying is prepared into avian encephalomyclitis virus inactivated vaccine.
The method of above-mentioned production avian encephalomyclitis virus inactivated vaccine, in described step a, cell line is young hamster kidney passage cell (BHK21), chick embryo fibroblast system (DF-1).Young hamster kidney passage cell (BHK21) screens through limiting dilution assay clone purification.
The method of above-mentioned production avian encephalomyclitis virus inactivated vaccine, in described step c and d, the inoculum concentration of avian encephalomyelitis chicken embryo tissue poison is 0.5% ~ 2%(V/V of cell growth medium), cell toxicant inoculum concentration is 1% ~ 3%(V/V of cell growth medium).
The method of above-mentioned production avian encephalomyclitis virus inactivated vaccine, in described step c and d, after young hamster kidney passage cell (BHK21) virus inoculation, cultivates 24h, transfers 33 DEG C to and cultivate 96h ~ 120h again for 37 DEG C.
The method of above-mentioned production avian encephalomyclitis virus inactivated vaccine, in described step c and d, after chick embryo fibroblast system (DF-1) virus inoculation, cultivates 120 h ~ 144 h for 37 DEG C.
The method of above-mentioned production avian encephalomyclitis virus inactivated vaccine, in described step b, the formula of cell growth medium is: volumn concentration 90 ~ 92% low sugar DMEM or DMEM/F12 liquid, 8 ~ 10% new-born calf serum, appropriate antibiotic, pH value is adjusted to 7.0-7.4.
The method of above-mentioned production avian encephalomyclitis virus inactivated vaccine, in described step c and d, the formula of cell maintenance medium is: volumn concentration 97 ~ 99% low sugar DMEM or DMEM/F12 liquid, 1 ~ 3% new-born calf serum, appropriate antibiotic, pH value is adjusted to 7.2-7.4.
Compared with prior art, the present invention has following beneficial effect:
1. in the present invention, substitute chick embryo culture avian encephalomyclitis virus by cell line, viral adaptability be good, height of tiring;
2. in the present invention, young hamster kidney passage cell (BHK21), through purification screening, improves the sensitivity of cell to virus, ensure that the state of cell is homogeneous, and difference between reducing batch, guarantees the stable of processing parameter and product quality;
3. in the present invention, during virus inoculation, adopt synchronous inocalation method, without the need to absorption, simplify the operation, save manpower;
4., in the present invention, after young hamster kidney passage cell (BHK21) virus inoculation 24h, reduce cultivation temperature, slowed down cell senescence speed, improves cell viability, improve virus titer;
5. in the present invention, a mass discrepancy is criticized little with cell line production avian encephalomyclitis virus inactivated vaccine, features such as there is production technology simple and stable, easy to operate, output is large, cost is low, exogenous virus pollution probability is little, possess industrialized great production feasibility and can amplification, there is good economic benefit and application prospect.
Detailed description of the invention
Be described in further detail the present invention below in conjunction with specific embodiment, wherein each embodiment is only for explaining technical scheme of the present invention and and non-limiting scope of patent protection of the present invention.
Embodiment 1 limiting dilution assay clone purification screening young hamster kidney passage cell (BHK21)
Young hamster kidney passage cell (BHK21), avian encephalomyclitis virus Van Roekel strain (AEV VR strain) are all purchased from China Veterinery Drug Inspection Office.
The formula of cell growth medium is volumn concentration is 92% low sugar DMEM liquid, 8% new-born calf serum, and adjustment pH value is 7.2;
A. monoclonal cell strain preparation
A. the limiting dilution of cell
Preparation BHK21 cell suspension, after cell counting, carries out gradient dilution to 10 with cell growth medium
1individual cell/ml, i.e. every 0.1ml 1 cell.
B. the clone of cell cultivates
The cell suspension that above-mentioned dilution is good is added in 96 porocyte culture plates, 0.1ml/ hole, basis of microscopic observation, discard non-single cell and the not good cell hole of cell state, be placed in 37 DEG C, 5% CO
2cultivate, every day, observation of cell growth conditions also changed liquid in time, treated that cell proliferation is cloning community, chose the cell hole that growth conditions is good, digest, make suspension with EDTA-pancreatin, repeat above-mentioned cloning process 1 ~ 2 time, until filter out monoclonal growth hole.
C. the amplification culture of monoclonal cell strain
Choose monoclonal growth hole, use EDTA-trypsinization, carry out amplification culture through 24 porocyte culture plates and Tissue Culture Flask first, cell culture, to some, carries out the foundation of the frozen of monoclonal cell and cell bank.
B. the screening of avian encephalomyclitis virus high-adaptability cell strain
A. BHK21 monoclonal cell strain inoculation avian encephalomyelitis chicken embryo tissue poison
By V:V 1% inoculum concentration by avian encephalomyclitis virus chicken embryo tissue poison inoculation BHK21 monoclonal cell strain, cultivate 94h for 37 DEG C, results virus liquid.After results virus liquid freeze thawing 3 times, inoculation BHK21 monoclonal cell, carries out successive transfer culture, connects biography 6 generation.
B.BHK21 monoclonal cell strain cultivates the EID of avian encephalomyclitis virus
50detect
Virus liquid sterile saline is carried out gradient dilution, inoculates 6 age in days SPF Embryo Gallus domesticus through yolk sac approach, put 37 DEG C and hatch 288h, judge.EID is calculated according to " People's Republic of China's veterinary drug allusion quotation " (three, 2010 versions) record method
50.
C. result
A. the screening of avian encephalomyclitis virus vaccine strain high-adaptability BHK21 monoclonal cell strain.
The strain of BHK21 monoclonal cell strain 32 is obtained by limiting dilution assay amplification culture.By EID after avian encephalomyclitis virus vaccine strain infection BHK21 monoclonal cell strain
50detection, filtering out 1 strain BHK21 monoclonal cell: the 26th strain, be avian encephalomyclitis virus vaccine strain high-adaptability BHK21 monoclonal cell strain, and label is B26.
B. avian encephalomyclitis virus vaccine strain infects the EID of BHK21 monoclonal cell strain
50.
Table 1 avian encephalomyclitis virus vaccine strain infects the EID of B26 monoclonal cell strain
50
| Generation | 1 generation | 2 generations | 3 generations | 4 generations | 5 generations | 6 generations |
| Tire (LogEID 50/ml） | 5.08 | 5.33 | 5.53 | 5.38 | 5.53 | 5.20 |
Infected the testing result (table 1) of B26 monoclonal cell strain from avian encephalomyclitis virus vaccine strain, the virus liquid of avian encephalomyclitis virus vaccine strain infection monoclonal cell strain results is tired and is up to 10
5.38~ 10
5.53eID
50/ ml.10 are tired with clone provirus liquid
4.53~ 10
4.87eID
50/ ml compares, and improves more than 0.5 titre, and the BHK21 monoclonal cell strain that screening obtains infects the EID of avian encephalomyclitis virus vaccine strain
50titre is significantly improved.
B26 young hamster kidney passage cell (BHK21) is deposited in China typical culture collection center on November 12nd, 2014, and preservation address is Hubei China province wuchang, wuhan district Luo Jia Shan preserving number is CCTCC NO:C2014209; Described cell is more common, and its feature description is omitted.
Embodiment 2 young hamster kidney passage cell (BHK21) produces avian encephalomyclitis virus inactivated vaccine
Cell is with the young hamster kidney passage cell (BHK21) of culture presevation in embodiment 1;
Plant poison with avian encephalomyclitis virus Van Roekel strain (AEV VR strain) in embodiment 1;
The formula of cell growth medium is volumn concentration 92% low sugar DMEM liquid, 8% new-born calf serum, and adjustment pH value is 7.3;
The formula of cell maintenance medium is volumn concentration 98% low sugar DMEM liquid, 2% new-born calf serum, and adjustment pH value is 7.4;
A. select young hamster kidney passage cell (BHK21) cell line as seedling cell;
B. the going down to posterity and cultivation of seedling cell
When young hamster kidney passage cell (BHK21) forms good monolayer, after EDTA-trypsinization, adding cell growth medium and be prepared into BHK21 cell suspension, going down to posterity or virus inoculation for continuing;
C. the breeding of production seed culture of viruses
By BHK21 cell suspension, subpackage Tissue Culture Flask, accesses avian encephalomyelitis chicken embryo tissue poison by V/V 1% simultaneously; Cultivate 24h, transfer 33 DEG C of cultivations to for 37 DEG C; Change cell maintenance medium when meeting malicious 72h, then continue to cultivate 72h, results culture, is generation cell toxicant.After generation cell toxicant freeze thawing 3 times, inoculation BHK21 cell suspension, carry out successive transfer culture, the culture of results is secondary cell toxicant.
Detection is tired, and it is 10 that generation cell toxicant is tired
6.08eID
50/ ml, it is 10 that secondary cell toxicant is tired
6.33eID
50/ ml;
D. production seedling venom
By BHK21 cell suspension, subpackage Tissue Culture Flask, accesses avian encephalomyclitis virus production seed culture of viruses (secondary cell toxicant) by V/V 1% simultaneously; Cultivate 24h, transfer 33 DEG C of cultivations to for 37 DEG C; Change cell maintenance medium when meeting malicious 72h, then continue to cultivate 72h, gather in the crops culture as seedling venom ,-15 DEG C of preservations.Seedling venom is tired after testing is 10
6.20eID
50/ ml.
E. vaccine preparation
Formaldehyde seedling venom being added (V/V) 0.3% carries out deactivation, 37 DEG C of deactivations 48 hours.Add (V/V) 4% Tween 80 to deactivation venom, be stirred for aqueous phase; In white oil, add class 80 of department by (V/V) 6%, adding aluminium stearate by (W/V) 2%, is oil phase after sterilizing; Carry out emulsifying according to the volume ratio 2:1 of oil phase and aqueous phase, get product after emulsifying vaccine, and by finished product vaccine quantitative separating.
F. safety verification
With SPF chicken 10 in 3 ~ 5 week age, every neck dorsal sc or chest muscle injection finished product vaccine 2 plumage part (1.0ml), observe 21, and all do not occur locally and whole body abnormal response, vaccine safety is up to the standards.
G. vaccine potency inspection
With SPF chicken 10 in 3 ~ 6 week age, every neck dorsal sc or chest muscle injection finished product vaccine 1 plumage part (0.5ml), after 28 days, together with contrast chicken 5, with AEV VR strain chicken embryo tissue poison intracerebral injection, every only 0.03 ~ 0.05ml(toxic amount is 10
3.20eID
50), observe 21.Result: matched group 5/5 is fallen ill, immune group 1/10 is fallen ill, and vaccine potency is up to the standards.
Embodiment 3 chick embryo fibroblast system (DF-1) produces avian encephalomyclitis virus inactivated vaccine
Chick embryo fibroblast system (DF-1), avian encephalomyclitis virus Van Roekel strain (AEV VR strain) are all purchased from China Veterinery Drug Inspection Office.
The formula of cell growth medium is volumn concentration is 90% DMEM/F12 liquid, 10% new-born calf serum, and adjustment pH value is 7.0;
The formula of cell dimension liquid is volumn concentration is 98% DMEM/F12 liquid, 2% new-born calf serum, and adjustment pH value is 7.3;
A. select chick embryo fibroblast system (DF-1) as seedling cell;
B. the going down to posterity and cultivation of seedling cell
When chick embryo fibroblast (DF-1) forms good monolayer, after EDTA-trypsinization, adding cell growth medium and be prepared into DF-1 cell suspension, going down to posterity or virus inoculation for continuing;
C. the breeding of production seed culture of viruses
By DF-1 cell suspension, subpackage Tissue Culture Flask, accesses avian encephalomyelitis chicken embryo tissue poison by V/V 2% simultaneously; 37 DEG C of cultivations, change cell maintenance medium when meeting malicious 72h, then continue to cultivate 72h, and results culture, is generation cell toxicant.After generation cell toxicant freeze thawing 3 times, inoculation BHK21 cell suspension, carry out successive transfer culture, the culture of results is secondary cell toxicant.
Detection is tired, and it is 10 that generation cell toxicant is tired
5.87eID
50/ ml, it is 10 that secondary cell toxicant is tired
6.20eID
50/ ml;
D. production seedling venom
By DF-1 cell suspension, subpackage Tissue Culture Flask, accesses avian encephalomyclitis virus production seed culture of viruses (secondary cell toxicant) by V/V 2% simultaneously, 37 DEG C of cultivations, change cell maintenance medium when meeting malicious 72h, then continue to cultivate 72h, gather in the crops culture as seedling venom ,-15 DEG C of preservations.Seedling venom is tired after testing is 10
6.02eID
50/ ml.
E. vaccine preparation
Formaldehyde seedling venom being added (V/V) 0.3% carries out deactivation, 37 DEG C of deactivations 48 hours.Add (V/V) 4% Tween 80 to deactivation venom, be stirred for aqueous phase; In white oil, add class 80 of department by (V/V) 6%, adding aluminium stearate by (W/V) 2%, is oil phase after sterilizing; Carry out emulsifying according to the volume ratio 2:1 of oil phase and aqueous phase, get product after emulsifying vaccine, and by finished product vaccine quantitative separating.
F. safety verification
With SPF chicken 10 in 3 ~ 5 week age, every neck dorsal sc or chest muscle injection finished product vaccine 2 plumage part (1.0ml), observe 21, and all do not occur locally and whole body abnormal response, vaccine safety is up to the standards.
G. vaccine potency inspection
With SPF chicken 10 in 3 ~ 6 week age, every neck dorsal sc or chest muscle injection finished product vaccine 1 plumage part (0.5ml), after 28 days, together with contrast chicken 5, with AEV VR strain chicken embryo tissue poison intracerebral injection, every only 0.03 ~ 0.05ml(toxic amount is 10
3.20eID
50), observe 21.Result: matched group 4/5 is fallen ill, immune group 2/10 is fallen ill, and vaccine potency is up to the standards.
Claims (7)
1. produce a method for avian encephalomyclitis virus inactivated vaccine, it is characterized in that: comprise the steps:
A. select cell line as seedling cell;
B. the going down to posterity and cultivation of seedling cell
When seedling cell forms good monolayer, after EDTA-trypsinization, adding cell growth medium and be prepared into cell suspension, going down to posterity or virus inoculation for continuing;
C. the breeding of production seed culture of viruses
By seedling cell suspension, subpackage Tissue Culture Flask, Simultaneous vaccination avian encephalomyelitis chicken embryo tissue poison, changes cell maintenance medium when cultivating 72h, continues cultivation 48 ~ 72h, and results culture is generation cell toxicant; Generation cell toxicant Secondary Culture is secondary cell toxicant; Get a generation, secondary cell toxicant as production seed culture of viruses;
D. production seedling venom
By seedling cell suspension, subpackage Tissue Culture Flask, accesses avian encephalomyclitis virus production seed culture of viruses simultaneously and cultivates, change cell maintenance medium, continue cultivation 48 ~ 72h during 72h, results culture ,-15 DEG C of preservations;
E. vaccine preparation
Add adjuvant by after the seedling venom deactivation of results, emulsifying is prepared into avian encephalomyclitis virus inactivated vaccine.
2. a kind of method of producing avian encephalomyclitis virus inactivated vaccine according to claim 1, is characterized in that, in step a, cell line is young hamster kidney passage cell (BHK21), chick embryo fibroblast system (DF-1); Young hamster kidney passage cell (BHK21) screens through limiting dilution assay clone purification.
3. a kind of method of producing avian encephalomyclitis virus inactivated vaccine according to claim 2, it is characterized in that, in step c and d, the inoculum concentration of avian encephalomyelitis chicken embryo tissue poison is 0.5% ~ 2%(V/V of cell growth medium), cell toxicant inoculum concentration is 1% ~ 3%(V/V of cell growth medium).
4. a kind of method of producing avian encephalomyclitis virus inactivated vaccine according to claim 3, is characterized in that, in step c and d, after young hamster kidney passage cell (BHK21) virus inoculation, cultivates 24h, transfers 33 DEG C to and cultivate 96h ~ 120h again for 37 DEG C.
5. a kind of method of producing avian encephalomyclitis virus inactivated vaccine according to claim 4, is characterized in that, in step c and d, after chick embryo fibroblast system (DF-1) virus inoculation, cultivates 120 h ~ 144 h for 37 DEG C.
6. a kind of method of producing avian encephalomyclitis virus inactivated vaccine according to claim 5, it is characterized in that, in step b, the formula of cell growth medium is: volumn concentration 90 ~ 92% low sugar DMEM or DMEM/F12 liquid, 8 ~ 10% new-born calf serum, appropriate antibiotic, pH value is adjusted to 7.0-7.4.
7. a kind of method of producing avian encephalomyclitis virus inactivated vaccine according to claim 6, it is characterized in that, in step c and d, the formula of cell maintenance medium is: volumn concentration 97 ~ 99% low sugar DMEM or DMEM/F12 liquid, 1 ~ 3% new-born calf serum, appropriate antibiotic, pH value is adjusted to 7.2-7.4.
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Cited By (5)
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| CN106119212A (en) * | 2016-07-01 | 2016-11-16 | 瑞普（保定）生物药业有限公司 | Aviadenovirus strain, inactivated vaccine and preparation method |
| CN106190991A (en) * | 2016-07-25 | 2016-12-07 | 江苏省农业科学院 | A kind of avian encephalomyclitis virus, inactivated vaccine and preparation method thereof |
| CN106177937A (en) * | 2016-07-25 | 2016-12-07 | 江苏省农业科学院 | Chicken egg drop syndrome and avian encephalomyelitis bivalent inactivated vaccine and preparation method thereof |
| CN106282130A (en) * | 2016-10-08 | 2017-01-04 | 江苏省农业科学院 | A kind of I group 4 type aviadenovirus, inactivated vaccine and preparation method thereof |
| CN113061583A (en) * | 2021-03-30 | 2021-07-02 | 天津瑞普生物技术股份有限公司 | Method for culturing avian encephalomyelitis virus by using primary cells of chicken bursa of Fabricius |
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Cited By (8)
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| CN106119212A (en) * | 2016-07-01 | 2016-11-16 | 瑞普（保定）生物药业有限公司 | Aviadenovirus strain, inactivated vaccine and preparation method |
| CN106119212B (en) * | 2016-07-01 | 2019-12-31 | 瑞普（保定）生物药业有限公司 | Fowl adenovirus strain, inactivated vaccine and preparation method |
| CN106190991A (en) * | 2016-07-25 | 2016-12-07 | 江苏省农业科学院 | A kind of avian encephalomyclitis virus, inactivated vaccine and preparation method thereof |
| CN106177937A (en) * | 2016-07-25 | 2016-12-07 | 江苏省农业科学院 | Chicken egg drop syndrome and avian encephalomyelitis bivalent inactivated vaccine and preparation method thereof |
| CN106190991B (en) * | 2016-07-25 | 2019-09-24 | 江苏省农业科学院 | A kind of avian encephalomyclitis virus, inactivated vaccine and preparation method thereof |
| CN106282130A (en) * | 2016-10-08 | 2017-01-04 | 江苏省农业科学院 | A kind of I group 4 type aviadenovirus, inactivated vaccine and preparation method thereof |
| CN106282130B (en) * | 2016-10-08 | 2019-07-12 | 江苏省农业科学院 | A kind of 4 type aviadenovirus of I group, inactivated vaccine and preparation method thereof |
| CN113061583A (en) * | 2021-03-30 | 2021-07-02 | 天津瑞普生物技术股份有限公司 | Method for culturing avian encephalomyelitis virus by using primary cells of chicken bursa of Fabricius |
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Application publication date: 20150610 |