CN103316355A - Pseudorabies live vaccine and preparation method thereof - Google Patents
Pseudorabies live vaccine and preparation method thereof Download PDFInfo
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- CN103316355A CN103316355A CN201310276995XA CN201310276995A CN103316355A CN 103316355 A CN103316355 A CN 103316355A CN 201310276995X A CN201310276995X A CN 201310276995XA CN 201310276995 A CN201310276995 A CN 201310276995A CN 103316355 A CN103316355 A CN 103316355A
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Abstract
The invention relates to a freeze-drying protective additive which is composed of 1 to 2% of NZ-Amine AS, 0.1 to 0.2% of monosodium glutamate, 5 to 10% of saccharose, 1 to 2% of M199, 0.1 to 0.3% of trehalose, 8 to 15% of hydrolyzed gelatin, and the balance of water. The freeze-drying protective additive is used for preparing a freeze-dried pseudorabies vaccine. A heat-resisting protective agent can minimize the damage of various physical and chemical factors of a product to virus activity during sub-packaging and freezing-drying processes, greatly reduces the titer loss of the virus before and after freezing and drying and keeps the loss of being not greater than 0.2 titer. A heat-resisting protective agent live vaccine is high in degree of resistance to aging, the loss ratio of the virus kept for 10 days under a condition of 37 DEG C is not greater than 0.5 titer; the heat-resisting protective agent live vaccine can be stored for 24 months at 2 to 8 DEG C, the titer of the vaccine is reduced a little, so that the problem that the vaccine in general transporting and storing needs strict cold chain condition, which leads to energy waste and inconvenient storage, can be solved effectively.
Description
Technical field
The invention belongs to the vaccine preparing technical field, be specially a kind of pseudorabies living vaccines and preparation method thereof.
Background technology
Freeze-dried live vaccine is one of main vaccine of prevention poultry infectious disease, but the freeze drying protectant prescription that present state intradermal vaccine enterprise commonly uses is simple, and protective value is poor; If preserve under 2～8 ℃ of conditions, storage life only has short 3～6 months, mostly need to preserve under-15 ℃ of conditions.This just requires vaccine to carry out under cryogenic conditions as far as possible in preservation, transportation and use procedure, otherwise tiring of vaccine will be had a greatly reduced quality.In production of vaccine, transportation and use procedure, if the control of the cold chain system of any one link is improper, hot environment will cause vaccine potency to descend, and finally causes pestilence because of immuning failure.Therefore, the development of freeze-dried type vaccine is imperative.
Summary of the invention
The purpose of this invention is to provide a kind of pseudorabies living vaccines and preparation method, namely a kind ofly can still can keep 2～8 ℃ of lower long preservation the freeze-dried type pseudorabies freeze dried vaccine of vaccine valence, thereby remedy the deficiencies in the prior art.
The present invention at first provides a kind of vaccine freeze-drying protective agent, and it is composed as follows: 1～2% NZ-Amine AS, 0.1～0.2% monosodium glutamate, 5～10% sucrose, 1～2% M199,0.1～0.3% trehalose, 8～15% gelatin hydrolysates, surplus are water.
The present invention also provides a kind of pseudorabies freeze dried vaccine, includes as the Pseudorabies virus of antigen and above-mentioned protective agent.
Also added penicillin and streptomycin in the above-mentioned pseudorabies freeze dried vaccine, its final concentration is respectively penicillin 100IU/ml, streptomycin 80 μ g/ml.
Above-mentioned lyophilizing freeze drying protectant, its preparation method is as follows:
1) at first take by weighing NZ-Amine AS, monosodium glutamate, sucrose, M199 culture medium dry powder and trehalose, filtrate is made in filtration sterilization after the dissolving; Employed microporous filter membrane aperture is 0.22 μ m.
2) take by weighing gelatin hydrolysate and join in the 70-80 ℃ of water for injection, stir until fully dissolving, autoclaving is made gelatin solution; The autoclaving condition is 116 ℃, 20min.
3) with filtrate and the step 2 of step 1) preparation) gelatin solution 4:1 mix after, make freeze drying protectant.
Freeze drying protectant of the present invention can reduce goods various chemical factors in packing and freeze-drying process to greatest extent makes the loss of tiring of lyophilizing front and back virus less to the damage of virus activity, is no more than 0.2 titre;
The ageing-resistant degree of freeze drying protectant live vaccine of the present invention is high; the loss rate of preserving 10 days virus under 37 ℃ of conditions is no more than 0.5 titre; under 2～8 ℃ of conditions, preserved 24 months; vaccine valence reduces less, and efficiently solving conventional vaccine needs strict cold chain condition and the energy waste that brings and the problem that stores inconvenience in transportation and preservation process.
The specific embodiment
In the freeze drying protectant preparation field of vaccine, the selection of protective agent component and proportioning have important impact to the preservation effect of vaccine.The applicant has carried out long-term research to the component of freeze drying protectant of the present invention, and has optimized matched proportion density to each other, has maximum composite effect between each component thereby make.
Be described below for the employed component of freeze drying protectant of the present invention:
1, NZ amine (NZ-Amine AS) is called again caseic enzymatic hydrolysis thing;
2, monopotassium glutamate salt: be a kind of surfactant, can reduce the tension force at interface, freeze with dehydration in can reduce ice, water termination tension force is caused freezes and the distortion of dewatering, and can play the wetting agent effect to active component in reconstitution process again.
3, sucrose has osmosis, can suppress the harmful microorganism growth, prolongs the product preservation term, has good water solublity.Can improve Microbial survival rate, form the homogeneous suspension, play the moisture mitigation, can prevent the active component degeneration.
4, M199 culture medium dry powder contains the nutritional labelings such as inorganic salt, aminoacid, vitamin and anhydrous glucose, Sodium Pyruvate, can be used for cell culture, and the present invention selects the dry powder form to use.
5, trehalose: (Trehalose) be a kind of disaccharidase that extensively exists in natural animals and plants and the microorganism, it is by α, α-1, the formed nonreducing sugar of 1-glycosidic bond by 2 glucoses;
6, gelatin hydrolysate can remove impurity albumen, no antigen, without anaphylaxis, without thermal source; and molecular weight is little, homogenizing, soluble in water; but filtration sterilization; eutectic point is-12 ℃; microorganism there is protective effect; can promote its distillation to form lyophilizing skeleton blocking-up conduction of heat and heat radiation, can prevent from that active substance from distilling to disperse and make it with steam to be shaped.Protective effect to microorganism exceeds common gelatin more than 10%.
Can select arbitrary money commercially available prod for the component of freeze drying protectant of the present invention, for example NZ-amine, monosodium glutamate, gelatin hydrolysate are all available from SIGMA company, and M199 culture medium dry powder, trehalose are available from GIBCO, and sucrose is available from Merck KGaA company.
The used pseudorabies virus of the present invention is selected existing Strain, for example Bathar-k61 strain.
The present invention is described in detail below in conjunction with specific embodiment.
One, the preparation of freeze drying protectant
Table 1: the formula table of each embodiment of lyophilizing freeze drying protectant (minute A liquid and B liquid two parts)
1) A liquid preparation: take by weighing respectively NZ-Amine AS, monopotassium glutamate salt, sucrose, M199 and trehalose, above composition is dissolved in the 100ml water for injection in order, fully shake up after the dissolving, with 0.22 μ m membrane filtration degerming, save backup in 37 ℃ of greenhouses.
2) B liquid preparation: after taking by weighing gelatin hydrolysate and fully dissolving with 70-80 ℃ 100ml water for injection, stir, 121 ℃, 15 pounds autoclavings 20 minutes are kept at after the sterilization in 37 ℃ of greenhouses and save backup.
3) by A liquid: the volume ratio of B liquid=1:1 carries out proportioning, and mix homogeneously is the lyophilizing freeze drying protectant.
Two, preparation pseudorabies freeze dried vaccine
1, the preparation of pseudorabies virus liquid
1. the recovery of PK-15 cell, cultivation: from liquid nitrogen, take out frozen PK-15 cell (available from U.S. ATCC cell bank) cryopreservation tube, be placed on rapidly and make its quick thawing in 37 ℃ of water-baths.With the cell transfer in each cryopreservation tube to the centrifuge tube that fills the 5ml cell growth medium, centrifugal 5 minutes of 500r/min, abandoning supernatant, with 10ml cell growth medium (the MEM nutritional solution that contains 10% calf serum, 1%L-glutamine, 100IU/ml penicillin, 80ug/ml streptomycin) re-suspended cell, cell is moved in the disposable import Tissue Culture Flask piping and druming evenly, put static cultivation in 37 ℃, 5%CO2 incubator.After forming good monolayer in 48～72 hours, discard growth-promoting media, wash the cell face 2 times with PBS, with cell dissociation buffer (0.25% pancreatin-0.02%EDTA) cell dissociation is got off, when treating that the slit of needle point size appears in the cell face, cell dissociation buffer is abandoned in suction, pats the cell bottle, and cell evenly comes off along the bottle wall, draw cell growth medium with suction pipe this moment, blow and beat gently cell dispersion, make cell be separated into individual cells, the cell suspension that disperses is carried out amplification culture in the ratio of 1:3～1:4.
2. virus propagation: when cell covers with monolayer, pseudorabies virus is inoculated on the cell monolayer, supply cell maintenance medium and (contain 2% calf serum in the MEM nutritional solution, 100IU/ml penicillin, 80ug/ml streptomycin, pH7.4～7.6) put 35 ℃～36 ℃ cultivations, every day the observation of cell pathological changes, when treating that cytopathy reaches 75%～80%, results pseudorabies virus culture fluid, freeze thawing once saves backup in-20 ℃ of freezers.
2, pseudorabies poisons steriling test and the viral level of results are measured; the a part of lyophilizing freeze drying protectant with preparing of the qualified virus liquid by volume ratio of 1:1 mixes (embodiment 1 vaccine group; wherein freeze drying protectant is composed as follows: 1% NZ-Amine AS, 0.1% monosodium glutamate, 5% sucrose, 1% M199,0.1% trehalose, 8% gelatin hydrolysate, surplus are that the concentration unit of stating waterborne is g/ml.Embodiment 2 vaccine group; it is composed as follows for freeze drying protectant: 2% NZ-Amine AS, 0.2% monosodium glutamate, 10% sucrose, 2% M199,0.3% trehalose, 15% gelatin hydrolysate; surplus is water); another part mixes with 5% sucrose milk protective agent; the two adds respectively penicillin and streptomycin in proportion, makes its final concentration be respectively penicillin 100IU/ml, streptomycin 80 μ g/ml, fully shakes up; quantitative separating carries out rapidly lyophilisation.The vaccine of cooing the protective agent lyophilizing of milk sucrose is the conventional vaccine matched group.
3, will divide the virus liquid that installs to pack in the lyophilizing cabinet, choose the pre-freeze temperature for-50 ℃, the low temperature retention time be that 2h, pre-freeze are controlled at-15 ℃ in earlier stage during pre-freeze, quick freezing after product discharges heat of desorption: the sublimation stage product temperature is 11h for-33 ℃, flaggy Temperature Setting for-8 ℃, distillation time; Desorption temperature is that 26 ℃, desorption time are 4h, and the lyophilizing overall process is 24h, and the outlet of jumping a queue obtains the freeze-dried type pseudorabies living vaccines.
The indices test of the freeze-drying prods of embodiment preparation
1, viral level is measured the finished product vaccine is diluted to 10 with cell maintenance medium before and after the lyophilizing
-4, 10
-5, 10
-6, 10
-74 dilution factors, inoculation has grown up to the PK-15 cell 96 porocyte plates of monolayer respectively, and each dilution factor is inoculated 6 holes, and every hole 0.1ml is containing 5%CO
2, incubator was cultivated 5～7 under 37 ℃ of conditions, calculated TCID with the Reed-Muench method
50, every part viral level answers 〉=10
4TCID
50, make comparisons with the matched group viral level simultaneously, the results are shown in Table 2.
Table 2: viral level (TCID before and after the vaccine freeze-drying of preparation of the present invention
50/ head part) changes
| Group | Before the lyophilizing | After the lyophilizing |
| Embodiment 1 vaccine group | 10 4.5 | 10 4.4 |
| Embodiment 2 vaccine group | 10 4.4 | 10 4.3 |
| The conventional vaccine matched group | 10 4.5 | 10 4.2 |
Result of the test shows, adopts the pseudorabies living vaccines lyophilizing front and back viral level loss of freeze drying protectant preparation of the present invention few, reduces by 0.1 titre; And larger with the conventional vaccine viral level loss of milk sucrose lyophilizing, be 0.3 titre.Illustrate that freeze drying protectant of the present invention has better protective effect than the GPF (General Protection False agent to Pseudorabies virus.
2, the of storage test is placed the vaccine for preparing among three embodiment respectively under 37 ℃, 2～8 ℃ ,-15 ℃ conditions together with existing conventional vaccine and is preserved, and regularly takes out several bottles, measures its character, vacuum, residual moisture and viral level.Detailed results sees Table 3.
Table 3: preserve different time character, vacuum, residual moisture, bioactivity result for 37 ℃
Result of the test shows, the pseudorabies living vaccines that adopts freeze drying protectant lyophilizing of the present invention 37 ℃ 7 days, tire and reduce by 0.1 titre, conventional Seedling is tired and is reduced by 0.5 titre; Preserved 10, freeze-dried vaccine is tired to reduce and is no more than 0.3 titre, and conventional Seedling is tired and reduced by 0.9 titre.As seen freeze drying protectant has better protection effect than GPF (General Protection False agent to virus.
Table 4: 2～8 ℃ of preservations of lyophilizing sample different time character, vacuum, residual moisture, bioactivity result
Result of the test shows, freeze-dried vaccine of the present invention is put 2～8 ℃ and preserved 12 months, and tiring to reduce is no more than 0.2 titre, and conventional Seedling is tired and reduced by 0.4 titre; Preserved 18 months, tiring to reduce is no more than 0.3 titre, and conventional Seedling reduces by 0.7 titre; Preserved 24 months, tiring to reduce is no more than 0.5 titre, and conventional Seedling is tired and reduced by 1.2 titres.
Table 5: freeze dried vaccine is preserved different time character, vacuum, residual moisture, bioactivity result at-15 ℃
Result of the test shows, freeze-dried vaccine of the present invention is positioned over-15 ℃ and preserved 12 months, tire almost constant, and conventional Seedling tired and reduced by 0.2 titre; Preserved 18 months, freeze-dried vaccine is tired to reduce and is no more than 0.2 titre, and conventional Seedling is tired and reduced by 0.4 titre; Preserved 24 months, freeze-dried vaccine is tired to reduce and is no more than 0.3 titre, and existing conventional Seedling is tired and reduced by 0.7 titre.
Can find out from above result of the test, lyophilizing freeze drying protectant of the present invention has good protective effect to pseudorabies virus, is better than conventional vaccine with common protective agent lyophilizing from character, residual moisture, vacuum and the several aspects protection successfuls of tiring.
3, the safety testing of vaccine with 6～18 monthly ages without 8 of the sheep of pseudorabies virus neutralizing antibody; be divided at random 2 groups; every group 4; heavy dose of intramuscular injection is stored in 2～8 ℃ of frozen-dried protective vaccinating agent of the present invention and matched group conventional vaccines of 24 months respectively; 5ml/ head (14 parts); another group injection physiological saline solution, the 5ml/ head.Observe after the injection and have or not the anaphylaxis such as erubescence, adnormal respiration occur, spit out white foams, observe every day and record sheep searches for food, drinks water, spirit, whether normal by hair and injection site, Continuous Observation 14 days.The results detailed in Table 6.
Table 6: the vaccine of preparation is stored in 2～8 ℃ of two kinds of vaccines of 24 months to sheep safety testing result
Annotate: " 4/5 is normal " represents that injection conventional vaccine matched group has 1 sheep to occur searching for food and reduces slight anaphylaxis, recover voluntarily after 3 days.
Result of the test shows, the vaccine of freeze drying protectant lyophilizing of the present invention is good to the safety of sheep, and is little to the animal zest, without any anaphylaxis, obviously is better than the conventional vaccine matched group.
4, Vaccine potency test (Immunization protection test) is with 6～18 monthly ages, without 12 of the sheep of Pseudorabies virus neutralizing antibody; be divided at random; 3 groups, 4 every group, intramuscular injection is stored in 2～8 ℃ of freeze dried vaccines of the present invention of 24 months and each 1ml(0.2 head part of conventional vaccine respectively).Inoculate rear 14 days, every strong malicious 1ml(of each intramuscular injection pseudorabies of sheep contains 10
3.0LD
50), every injection of another group 1ml normal saline in contrast, with isolated rearing under the condition, Continuous Observation 14 days is searched for food behind every day entry sheep counteracting toxic substances, drinking-water, mental status, records dead quantity.
Result of the test is searched for food behind 4 sheep counteracting toxic substances of vaccine group of the present invention, is drunk water, spirit is all normal, and 4 sheep are strong living all, and protective rate is 100%; There are 2 sheep appetite to occur behind the conventional vaccine group sheep counteracting toxic substances and descend, lassitude, last dead, protective rate only is 50%; The equal appetite of 4 sheep of normal saline matched group descends, the food that almost gives up, and lethargy, the vertical or sleeping ground that often stands against the wall, dead 3, survival sheep loses weight.The results detailed in Table 7.
Table 7: vaccine is stored in 2～8 ℃ of two kinds of vaccines of 24 months to the sheep challenge test result of sheep
Result of the test shows, preserves 24 months under 2～8 ℃ of conditions with the pseudorabies living vaccines of freeze drying protectant lyophilizing of the present invention, still keeps good immunogenicity, and susceptible animal is had good protective effect, can effectively resist the attack of strong poison.Compare with conventional vaccine and to have obvious advantage.
Freeze drying protectant of the present invention can be protected the activity of virus well, has the incomparable superiority of GPF (General Protection False agent, has good development prospect.
Claims (5)
1. the freeze drying protectant of a vaccine, it is composed as follows: 1～2% NZ-Amine AS, 0.1～0.2% monosodium glutamate, 5～10% sucrose, 1～2% M199,0.1～0.3% trehalose, 8～15% gelatin hydrolysates, surplus are water.
2. freeze drying protectant claimed in claim 1, its preparation method is as follows:
1) at first take by weighing NZ-Amine AS, monosodium glutamate, sucrose, M199 and trehalose, filtrate is made in filtration sterilization after the dissolving; Employed microporous filter membrane aperture is 0.22 μ m;
2) take by weighing gelatin hydrolysate and join in the 70-80 ℃ of water for injection, stir until fully dissolving, autoclaving is made gelatin solution; The autoclaving condition is 116 ℃, 20min;
3) with filtrate and the step 2 of step 1) preparation) gelatin solution 4:1 mix after, make freeze drying protectant.
3. pseudorabies freeze dried vaccine, described vaccine includes as the Pseudorabies virus of antigen and freeze drying protectant claimed in claim 1.
4. pseudorabies freeze dried vaccine claimed in claim 3 is characterized in that, also adds penicillin and streptomycin, and its final concentration is respectively penicillin 100IU/ml, streptomycin 80 μ g/ml.
5. pseudorabies freeze dried vaccine claimed in claim 3 is used for the prevention pseudorabies.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109758427A (en) * | 2019-02-26 | 2019-05-17 | 福州大北农生物技术有限公司 | A kind of pseudorabies living vaccines heat resisting protective and its preparation method and application |
| CN111961592A (en) * | 2020-08-04 | 2020-11-20 | 中牧实业股份有限公司 | RNA virus preservation solution and preparation method and application thereof |
| CN113813374A (en) * | 2021-09-09 | 2021-12-21 | 豪威生物科技有限公司 | Heat-resistant freeze-drying protective agent for avian adenovirus virus seeds and preparation method thereof |
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| US6656478B1 (en) * | 1999-11-12 | 2003-12-02 | Samuel D. Charles | Cross-protective salmonella vaccines |
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| US6656478B1 (en) * | 1999-11-12 | 2003-12-02 | Samuel D. Charles | Cross-protective salmonella vaccines |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109758427A (en) * | 2019-02-26 | 2019-05-17 | 福州大北农生物技术有限公司 | A kind of pseudorabies living vaccines heat resisting protective and its preparation method and application |
| CN109758427B (en) * | 2019-02-26 | 2021-11-02 | 兆丰华生物科技(福州)有限公司 | Pseudorabies live vaccine heat-resistant protective agent and preparation method and application thereof |
| CN111961592A (en) * | 2020-08-04 | 2020-11-20 | 中牧实业股份有限公司 | RNA virus preservation solution and preparation method and application thereof |
| CN113813374A (en) * | 2021-09-09 | 2021-12-21 | 豪威生物科技有限公司 | Heat-resistant freeze-drying protective agent for avian adenovirus virus seeds and preparation method thereof |
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