CN106754762A - A kind of antigen of encephalitis B live vaccine and preparation method and application - Google Patents
A kind of antigen of encephalitis B live vaccine and preparation method and application Download PDFInfo
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Abstract
The present invention relates to animal vaccine, a kind of antigen of encephalitis B live vaccine and preparation method and application is specifically disclosed.The spinner culture of japanese encephalitis virus attenuated strain (14 2 plants of SA14) is carried out using Vero cells, the Virus culture maintaining liquid containing compositions such as 4 HEPESs, arginine monohydrochloride, PEG2000 is used, produce the antigen for obtaining, with differences between batches are small, the advantage such as immunogenicity is good, malicious valency is high, the japanese encephalitis virus of pig can be effectively prevented to infect with pig japanese b encephalitis live vaccine prepared by the antigen.
Description
Technical field
The present invention relates to animal vaccine, specifically, it is related to the antigen and the epidemic disease containing it of a kind of encephalitis B live vaccine
Seedling.
Background technology
The antigen mode of production of existing pig Vaccinum Encephalitis B includes two kinds, and one kind is using the strong poison inoculation of encephalitis B
Mouse, collects infecting mouse brain tissue, and through the antigen that tissue homogenate is prepared from, it is high, miscellaneous that the method has bio-safety risk
Albumen is more, the problems such as be unsuitable for large-scale production；Another kind is, using primary hamster kidney cell spinner culture vaccine low virulent strain, to be somebody's turn to do
Method has that primary cell preparation technology is complicated, exogenous virus control is difficult, differences between batches, especially cultivates virus poison
Valency is low, is only capable of reaching 5.5-7.0LgPFU/mL, increased the production cost of vaccine.
The content of the invention
In order to solve problems of the prior art, it is an object of the invention to provide a kind of the anti-of encephalitis B live vaccine
Original and preparation method and application.
In order to realize the object of the invention, technical scheme is as follows：
In a first aspect, the invention provides a kind of antigen of encephalitis B live vaccine, its preparation method comprises the following steps：
1) japanese encephalitis virus 0.02~0.2MOI of the attenuated strain (PFU/ for having adapted to Vero cells are inoculated with Vero cells
Cell number), adsorbed；
2) the Virus culture maintaining liquid of addition pH7.6~7.8 is cultivated；
The Virus culture maintaining liquid be the calf serum containing 15~20mL/L, 6~15g/L arginine monohydrochlorides, 5~
10g/L PEG2000 and 2.3~12g/L 4- HEPESs, the DMEM solution of pure water configuration；
3) treat that 60~80% lesions occur in vero cells, freeze thawing harvests virus liquid, and clarification filtration removes cell fragment, collects
Viral supernatant liquid, obtains final product.
Preferably, the pH value of the Virus culture maintaining liquid is 7.6, adjusted using the NaOH of 1M, the Virus culture
Maintaining liquid through membrane filtration it is degerming after use.
Preferably, the Virus culture maintaining liquid be the calf serum containing 20mL/L, 10g/L arginine monohydrochlorides,
5g/L PEG2000 and 5g/L4- HEPESs, the DMEM solution of pure water configuration.
Preferably, step 1) in, under conditions of 37 DEG C, 12~20 turns/hour, adsorb 1h.Step 2) in, 35
DEG C, under conditions of 12~20 turns/hour, cultivate 3~4 days.Above-mentioned condition, can advantageously in viruses adsorption and stabilization.
Further, step 1) in, the preparation method of the Vero cells for being inoculated with attenuated strain is：Using containing volume basis
Than the DMEM cell culture fluid spinner culture Vero cells of 8% calf serum, treat that Vero cells form individual layer, discard cell culture
Liquid, with the PBS 3 times of pH7.4, discards washing lotion, obtains final product.
Further, step 1) in, the japanese encephalitis virus attenuated strain for having adapted to Vero cells is through Vero cells
After Secondary Culture, malicious valency is more than 7LgPFU/mL, and the less attenuated strain of passage number.
Preferably, the japanese encephalitis virus attenuated strain for carrying out Vero passage cultures is SA14-14-2 plants, it is purchased from
The pig japanese b encephalitis live vaccine (SA14-14-2 plants) of Zhongmu Industry Co., Ltd's sale, be through Vero passage cultures
Can obtain.
Second aspect, the application the invention provides aforementioned antigens in Vaccinum Encephalitis B is prepared.
It should be noted that the vaccine can be carried out using the conventional vaccine preparation method in this area, but as long as using
Antigen of the present invention carries out the preparation of vaccine, belongs to protection scope of the present invention.
The third aspect, the invention provides a kind of encephalitis B live vaccine of the pig containing aforementioned antigens.
Further, prepared through vacuum freeze drying after it is mixed by the antigen with freeze drying protectant.Every part virus
Content is not less than 105.0PFU。
The program of the vacuum freeze drying is：In -40 DEG C of pre-freeze stage, vaccine to reach that hold time after minimum temperature 2 small
When；- 20 DEG C of lyophilization stage vaccines final temperature, run time 17 hours when reaching vaccine final temperature, freeze drying box vacuum
Control pressure is 0.18mbar；28 DEG C of the final temperature of parsing-desiccation stage vaccines, freeze drying box vacuum pressure is 0.005mbar,
Run time 8 hours.
The beneficial effects of the present invention are：
The invention provides a kind of antigen of encephalitis B live vaccine and preparation method and application, entered using Vero cells
The spinner culture of row japanese encephalitis virus attenuated strain (SA14-14-2 plants), has used and has contained 4- HEPESs, smart ammonia
The Virus culture maintaining liquid of the compositions such as acid hydrochloride, PEG2000, produces the antigen for obtaining, with differences between batches are small, immunogene
Property the advantage such as good, malicious valency is high, the pig japanese b encephalitis live vaccine prepared with the antigen can effectively prevent the japanese encephalitis virus sense of pig
Dye.
Specific embodiment
The present invention is further described with reference to specific implementation example, advantages of the present invention and feature will be with description
And it is apparent.But these examples of implementation are only exemplary, do not constitute any limitation to the scope of the present invention.This area skill
Art personnel should be understood that without departing from the spirit and scope of the invention can be to the details of technical solution of the present invention and shape
Formula is modified or is replaced, but these modifications and replacement are each fallen within protection scope of the present invention.
Embodiment 1 adapts to the preparation and immunogenicity research of Vero cell encephalitis Bs virus attenuation strain
(1) viral adaptability passage
Vero cell suspensions are prepared with the DMEM cell culture fluids containing 8% calf serum, according to point kind of a rate 1：3 inoculated and cultureds
Bottle, 37 DEG C, 5%CO2Culture, treats that cell forms fine and close individual layer (about cultivating 48 hours), for connecing poison.Connect in blake bottle before poison
Cell culture fluid evacuation, with the PBS 3 times of pH7.4, last time is net by raffinate control after washing, then according to nutrient solution
5% inoculation japanese encephalitis virus attenuated strain (SA14-14-2) of volume.Poison is connect to be adsorbed 90 minutes after 37 DEG C.Absorption is finished, plus
Enter containing 2% calf serum DMEM maintaining liquids, in 35 DEG C of cultures.Day by day observation of cell lesion situation, when cytopathy reaches 75%
When terminate culture, virus is harvested after freeze thawing and is denoted as F1 and is treated, according to said method by the generation of Japanese encephalitis attenuated virus strain continuous passage 10, determine each
Generation poison valency.
(2) measure of viral titer
It is serially diluted using carrying out 10 times to each generation virus liquid containing 2% calf serum DMEM, inoculation 24 porocyte plates training
Foster BHK-21 cell monolayers, 0.1mL/ holes, 37 DEG C, 5%CO260min is adsorbed in incubator, every 30 minutes jog plates 1 time.
Be inverted for culture plate after terminating by absorption, drains remaining venom in hole, is then added per hole and contains 1.5% methylcellulose, 2% calf
The DMEM of serum maintains culture medium 2mL, covers cell monolayer, inserts 37 DEG C, 5%CO2Incubator continues to cultivate to be carried out for 5~6 days
Fixed dyeing, is added per hole and contains 1% crystal violet, and the fixed coloring agent 0.5mL of 20% methyl alcohol, room temperature is placed 20 minutes, then abandoned
Coloring agent is removed, is rinsed 1 time with clear water, it is drained, put culture plate and dry in the shade in drying ventilation.On fixed staining cell plate just
Normal cell color is in bluish violet, and lesion region cell is not colored or for light blue.The erosion in each hole is counted under low-powered microscope mirror
Spot number, calculates the average plaque number (PFU) of certain dilution factor, and the plaque content of detection sample is calculated according to formula.Sample loses
Average plaque number × extension rate × 10 of spot content (LgPFU/mL)=Lg dilution factors.
As a result, japanese encephalitis virus attenuated strain SA14-14-2 is progressively adapted to after continuous passage on Vero cells, malicious valency
The trend increased by generation is presented, in F8-F10 stabilizations to be achieved, is attenuated the virus liquid in the range of this generation as encephalitis B virus
Seed culture of viruses prepared by strain antigen.
Table 1：The malicious valency change of adaptability passage of the japanese encephalitis virus attenuated strain on Vero cells
(3) the pure property and Study On Immunogenicity of seed culture of viruses
Pure property inspection：Reference《Republic of China Veterinary Pharmacopoeia》Method carries out steriling test, mycoplasma and checks and outer
Source virus examination.
Immunogenicity experiments：Select F8, F9, F10 of Vero cell culture thin for encephalitis B virus liquid and primary suslik kidney
The encephalitis B virus liquid (being denoted as PHK-SA14-14-2) of born of the same parents' culture, carries out 10 times and is serially diluted, often for seed culture of viruses with 2% serum DMEM
Take 10- 2、10- 3With 10- 4Three dilution factors, every dilution factor 10~12g of subcutaneous vaccination small white mouses 10,0.1mL/ only, is immunized one
Secondary, whole small white mouses are attacked with the strong P3 plants of abdominal cavity of poison of encephalitis B on the 14th after immune, every mice by intraperitoneal injection 0.3mL
Every empty thorn of intracerebral simultaneously is attacked in (being not less than 500*LD50), abdominal cavity, destroys blood-brain barrier, separately sets same batch control small white mouse 10
Only, it is not immunized, but carry out attacking poison together with immune group.Attack after poison that death is disregarded in 3 days, remaining observation 14 days, control group
Encephalitis syndrome should occur in small white mouse, and more than 80% is dead, and each immune group median effective dose (ED50) is calculated after 14 days.According to
The median effective dose of each immune group calculates median protective dose, median protective dose=seed culture of viruses poison valency × half effective agent
Amount.
As a result, F8, F9, F10 are dirty without bacterium, mould, mycoplasma and exogenous virus for Vero cell culture encephalitis B virus liquid
Dye.Immunogenicity is suitable with the viral median protective dose of primary hamster kidney cell culture, meets and the half of small white mouse is protected
Protect the requirement of dosage≤1000PFU.
Table 2：The pure property and Study On Immunogenicity result of japanese encephalitis virus attenuated strain
| Sequence number | Viral species | Steriling test | Mycoplasma is checked | Exogenous virus is checked | Median effective dose |
| 1 | F8 | It is negative | It is negative | It is negative | 19 |
| 2 | F9 | It is negative | It is negative | It is negative | 21 |
| 3 | F10 | It is negative | It is negative | It is negative | 19 |
| 4 | PHK-SA14-14-2 | It is negative | It is negative | It is negative | 21 |
Embodiment 2 prepares encephalitis B virus of live vaccine liquid (antigen) using 3L rolling bottles
(1) preparation of antigen
Technical scheme 1：Using 3L spinner culture Vero, culture medium is the DMEM cell culture fluids containing 8% calf serum, is treated
Vero cells form individual layer, discard cell culture fluid, with the PBS 3 times of pH7.4, discard PBS washing lotions；Inoculation adapts to Vero
The F8 of cell for japanese encephalitis virus attenuated strain (SA14-14-2 plants) 0.2MOI (PFU/ cell numbers), Rotary Machine revolution is 12 turns/
Hour, 37 DEG C of absorption 1h；Addition 0.22ul membrane filtrations degerming pH7.6 DMEM solution (calf serum containing 20mL/L,
10g/L arginine monohydrochlorides, 5g/L PEG2000 and 5g/L 4- HEPESs), it is incubated under the conditions of 35 DEG C,
Rotary Machine revolution is 12 turns/hour；There is 75% lesion in about 3~4d, cell, and freeze thawing once harvests virus liquid, clarified elimination
Fall cell fragment, collect viral supernatant liquid.According to said method prepare vaccine antigen 3 batches.
Technical scheme 2：Viral maintaining liquid contains 2% calf serum from the degerming rear pH7.6 of 0.22ul membrane filtrations
DMEM solution, other according to said method prepare vaccine antigen 3 batches with technical scheme 1.
Technical scheme 3：According to the production of (SA14-14-2 plants) of pig japanese b encephalitis live vaccine and inspection procedure, using primary
Hamster kidney cell production encephalitis B live vaccine antigen 3 batches.
(2) measure of malicious valency
The poison valency measures method of reference implementation example 1 is carried out.As a result, the 3 batches of antigens poison valency for being prepared with technical scheme 1 reaches respectively
To 7.90,8.04,7.85LgPFU/mL, each batch poison price differential is different smaller；Technical scheme 2, employs conventional viral nutrient solution, system
Standby 3 batches of antigens poison valency can also reach more than 7.2LgPFU/mL；Technical scheme 3, using the anti-of primary hamster kidney cell culture
Former poison valency is relatively low, and differences between batches are larger, it can be seen that the effect of technical scheme 1 is best.
(3) pure property inspection
Reference《Republic of China Veterinary Pharmacopoeia》Method carries out steriling test, mycoplasma inspection and exogenous virus inspection.
As a result, the 9 batches of antigens for being prepared using technical scheme 1, technical scheme 2, technical scheme 3, without bacterium, mould, mycoplasma, external source
Virus pollution.
Table 3：3L spinner culture encephalitis B live vaccines antigen poison valency and pure property testing result
Embodiment 3 prepares pig encephalitis B live vaccine using 15L rolling bottles
(1) preparation of antigen
Using 15L spinner culture Vero cells, culture medium is the DMEM cell culture fluids containing 8% calf serum, treats Vero
Cell forms individual layer, discards cell culture fluid, with the PBS 3 times of pH7.4, discards PBS washing lotions；It is inoculated with the F8 of Vero cells
For japanese encephalitis virus attenuated strain (SA14-14-2 plants) 0.02MOI (PFU/ cell numbers), Rotary Machine revolution is 20 turns/hour, 37
DEG C absorption 1h；Add pH7.8 Virus culture maintaining liquid DMEM (containing the calf serum of 20mL/L, 10g/L arginine monohydrochlorides,
5g/L PEG2000 and 5g/L 4- HEPESs), it is incubated under the conditions of 35 DEG C, Rotary Machine revolution be 20 turns/it is small
When；Treat that 75% lesion occurs in cell, freeze thawing once harvests virus liquid, and clarification filtration removes cell fragment, collects viral supernatant liquid.
According to said method prepare Vaccinum Encephalitidis Epidemicae antigen 3 batches.
(2) measure of antigen poison valency
Antigen poison valency measures method with embodiment 1 poison valency measures method.As a result, 3 batches of antigens poison valency of preparation reaches respectively
To 8.00,7.95,7.76LgPFU/mL.
(3) safety examination of antigen
Method 1：Subcutaneous infection pathogenicity
By viral antigen 10~12g of subcutaneous vaccination small white mouses 10, every injection 0.1mL, while carrying out the empty thorn of intracerebral, 3
In a few days death toll must not exceed 2, and remaining small white mouse continues to observe 14, should all be good for work.
Method 2：By viral antigen with 107.0It is negative that the dosage posterior auricular muscle meat of PFU injects 40~45 age in days encephalitis neutralizing antibodies
Piglet 4, observes 14, and piglet should have no adverse reaction.Each batch antigen inoculation mouse of result is all strong to live, and inoculation piglet is invariably
Good reaction.
Table 4：15L spinner culture encephalitis B live vaccines antigen poison valency and safety examination result
(4) preparation of vaccine and poison valency measures
(protective agent component is gelatin to 1506001 batches of vaccine antigens prepared by 15L rolling bottle Vero cells with freeze drying protectant
12g/L, sucrose 100g/L, trehalose 50g/L, PVP (K90) 2.5g/L, mannitol 5g/L, sodium glutamate 10g/L, are completely dissolved
Pure water is added afterwards to 1L, and adjusts pH value to 7.5 with the hydrochloric acid of 1N, through 116 DEG C, 30 minutes high pressure steam sterilizations) according to 1:1 mixes
Close, in packing to 7mL green grass or young crops bottles, every bottle of 2mL.Vacuum freeze drying is carried out, lyophilized program is：In -40 DEG C of pre-freeze stage, vaccine reaches
Held time after to minimum temperature 2 hours；- 20 DEG C of lyophilization stage vaccines final temperature, reaches vaccine final temperature luck
17 hours row time, freeze drying box vacuum control pressure is 0.18mbar；28 DEG C of the final temperature of parsing-desiccation stage vaccines, freezes
Case vacuum pressure is 0.005mbar, run time 8 hours.Roll lid sealing.Vaccine is named as pig japanese b encephalitis passage cell work epidemic disease
Seedling, lot number is CP-150601.
Poison valency measures are carried out after freeze dried vaccine is dissolved using sterile pure water, method is with embodiment 1.As a result, after conversion
Freeze dried vaccine poison valency is 7.80LgPFU/mL, and freeze-drying process vaccine antigen poison valency loses smaller, only 0.2LgPFU/mL.
(5) safety experiment of the vaccine to pig
40-45 ages in days JE neutralizing antibody feminine gender piglet 8 is divided into 2 groups every group 4, and one group is immune group, musculi colli inoculation
Pig japanese b encephalitis passage cell live vaccine (lot number：CP-150601), dosage of inoculation is 107.0PFU/ heads；Another group is control group,
Musculi colli injects pH7.4PBS1mL.Observed 14 days after inoculation, body temperature is measured 1 time before every morning feeding；Inoculation 0,1,2,
3 days collection blood plasma, uses《GB 22333-2008-T Japanese B encephalitis virus reverse transcriptional PCR test methods》Survey
Determine encephalitis B virus mass formed by blood stasis.
As a result, 4 pigs of pig japanese b encephalitis passage cell live vaccine, clinical symptoms without exception in the observation period, injection are inoculated with
Position NIP lesion, body temperature is normal, viremia virusemia does not occur, illustrates vaccine safety.
(6) Study On Immunogenicity of the vaccine to pig
40-45 ages in days JE neutralizing antibody feminine gender piglet 10 is divided into two groups of A, B, and every group 5, A groups are immune group, musculi colli
It is inoculated with the pig japanese b encephalitis passage cell live vaccine (lot number of dilution：CP-150601) 5.0LgPFU/mL, every injection 1mL；B groups
It is control group, intramuscular inoculation pH7.4PBS 1mL.28 days after inoculation, two groups of animals, every hypodermic injection 5 × 106LD50Encephalitis disease
Malicious strong P3 plants of poison, gathers all piglet plasma samples in the 1st, 2,3 days after poison is attacked, and uses《GB 22333-2008-T Japanese Bs
Encephalitis viruses reverse transcriptional PCR test method》Encephalitis B virus mass formed by blood stasis is determined, any of which day detects viremia virusemia
It is judged to infection and encephalitis B virus infection.
As a result, inoculation 5.0LgPFU pig japanese b encephalitis passage cells live vaccine can protect attacking for the pig resistance strong poison of encephalitis
Hit, illustrate that the vaccine has preferable Immunization protective effect to pig.
Table 5：Piglet immunological challenge test viremia virusemia detects situation
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (10)
1. a kind of antigen of encephalitis B live vaccine, it is characterised in that its preparation method comprises the following steps：
1) japanese encephalitis virus attenuated strain 0.02~0.2MOI (the PFU/ cells for having adapted to Vero cells are inoculated with Vero cells
Number), adsorbed；
2) the Virus culture maintaining liquid of addition pH7.6~7.8 is cultivated；
The Virus culture maintaining liquid is the calf serum containing 15~20mL/L, 6~15g/L arginine monohydrochlorides, 5~10g/
L PEG2000 and 2.3~12g/L4- HEPESs, the DMEM solution of pure water configuration；
3) treat that 60~80% lesions occur in Vero cells, freeze thawing harvests virus liquid, and clarification filtration removes cell fragment, collects supernatant
Virus liquid, obtains final product.
2. antigen according to claim 1, it is characterised in that the Virus culture maintaining liquid is the calf containing 20mL/L
Serum, 10g/L arginine monohydrochlorides, 5g/L PEG2000 and 5g/L 4- HEPESs, the DMEM of pure water configuration are molten
Liquid.
3. antigen according to claim 1 and 2, it is characterised in that step 1) in, in 37 DEG C, the bar of 12~20 turns/hour
Under part, 1h is adsorbed.
4. antigen according to claim 1 and 2, it is characterised in that step 2) in, in 35 DEG C, the bar of 12~20 turns/hour
Under part, cultivate 3~4 days.
5. antigen according to claim 1 and 2, it is characterised in that step 1) in, the Vero cells for being inoculated with attenuated strain
Preparation method be：Using the DMEM cell culture fluid spinner culture Vero cells containing the calf serum of percent by volume 8%, treat
Vero cells form individual layer, discard cell culture fluid, with the PBS 3 times of pH7.4, discard washing lotion, obtain final product.
6. antigen according to claim 1 and 2, it is characterised in that step 1) in, it is described to have adapted to the B-mode of Vero cells
Encephalitis viruses attenuated strain is that after Vero passage cultures, malicious valency is more than 7LgPFU/mL, and the less attenuation of passage number
Strain.
7. application of the antigen described in any one of claim 1~6 in Vaccinum Encephalitis B is prepared.
8. boar encephalitis B live vaccine, it is characterised in that contain the antigen described in any one of claim 1~6.
9. vaccine according to claim 8, it is characterised in that its mixed with freeze drying protectant by the antigen after through vacuum
It is prepared by freeze-drying.Every part viral level is not less than 105.0PFU。
10. vaccine according to claim 9, it is characterised in that the program of the vacuum freeze drying is：The pre-freeze stage-
40 DEG C, vaccine is held time 2 hours after reaching minimum temperature；- 20 DEG C of lyophilization stage vaccines final temperature, reaches vaccine
Run time 17 hours during final temperature, freeze drying box vacuum control pressure is 0.18mbar；Parsing-desiccation stage vaccines it is final
28 DEG C of temperature, freeze drying box vacuum pressure is 0.005mbar, run time 8 hours.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111018970A (en) * | 2019-12-27 | 2020-04-17 | 中牧实业股份有限公司 | Specific positive serum for porcine encephalitis B virus and preparation method thereof |
| CN114209823A (en) * | 2022-01-23 | 2022-03-22 | 中牧实业股份有限公司 | Adjuvant for pig Japanese encephalitis live vaccine composition, composition and application |
| CN114209822A (en) * | 2022-01-23 | 2022-03-22 | 中牧实业股份有限公司 | Adjuvant for pig Japanese encephalitis inactivated vaccine composition, composition and application thereof |
| CN114272366A (en) * | 2021-12-04 | 2022-04-05 | 辽宁成大生物股份有限公司 | Method for preparing inactivated Japanese encephalitis vaccine and vaccine |
| CN114317414A (en) * | 2022-01-23 | 2022-04-12 | 中牧实业股份有限公司 | Full suspension cell strain of vero cell and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102327609A (en) * | 2010-08-27 | 2012-01-25 | 丽珠集团疫苗工程股份有限公司 | Production method of encephalitis B vaccine |
| CN103301452A (en) * | 2013-07-03 | 2013-09-18 | 青岛易邦生物工程有限公司 | Lyophilized vaccine for swine encephalitis B and preparation method thereof |
| CN103656661A (en) * | 2013-11-21 | 2014-03-26 | 中牧实业股份有限公司 | Porcine Japanese encephalitis live vaccine heat-resistant lyophilized protective agent as well as preparation method and application thereof |
| CN105688202A (en) * | 2016-03-03 | 2016-06-22 | 四川农业大学 | Japanese encephalitis (JE) vaccine composition and preparing method thereof |
-
2016
- 2016-11-22 CN CN201611046055.1A patent/CN106754762A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102327609A (en) * | 2010-08-27 | 2012-01-25 | 丽珠集团疫苗工程股份有限公司 | Production method of encephalitis B vaccine |
| CN103301452A (en) * | 2013-07-03 | 2013-09-18 | 青岛易邦生物工程有限公司 | Lyophilized vaccine for swine encephalitis B and preparation method thereof |
| CN103656661A (en) * | 2013-11-21 | 2014-03-26 | 中牧实业股份有限公司 | Porcine Japanese encephalitis live vaccine heat-resistant lyophilized protective agent as well as preparation method and application thereof |
| CN105688202A (en) * | 2016-03-03 | 2016-06-22 | 四川农业大学 | Japanese encephalitis (JE) vaccine composition and preparing method thereof |
Non-Patent Citations (6)
| Title |
|---|
| MASARU NAWA: ""Japanese Encephalitis Virus Infection in Vero Cells:The Involvement of Intracellular Acidic Vesicles in he Early Phase of Viral Infection Was Observed with the Treatment of a Specific Vacuolar Type H+-ATPase Inhibitor, Bafilomycin Al"", 《MICROBIOL.IMMUNOL.》 * |
| 刘岩 等: "乙型脑炎病毒在Vero细胞上的传代适应性及病毒种库的建立"", 《中国生物制品学杂志》 * |
| 刘建福 等: "《细胞工程》", 30 June 2014, 华中科技大学出版社 * |
| 姚智慧 等: ""流行性乙型脑炎Vero细胞纯化灭活疫苗的研究"", 《中华实验和临床病毒学杂志》 * |
| 沈强 等: ""乙型脑炎及其疫苗概况"", 《生物技术通报》 * |
| 鲍明升: "《细胞生物学实验与习题指导》", 30 April 2015, 中国科学技术大学出版社 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111018970A (en) * | 2019-12-27 | 2020-04-17 | 中牧实业股份有限公司 | Specific positive serum for porcine encephalitis B virus and preparation method thereof |
| CN111018970B (en) * | 2019-12-27 | 2021-09-14 | 中牧实业股份有限公司 | Specific positive serum for porcine encephalitis B virus and preparation method thereof |
| CN114272366A (en) * | 2021-12-04 | 2022-04-05 | 辽宁成大生物股份有限公司 | Method for preparing inactivated Japanese encephalitis vaccine and vaccine |
| CN114272366B (en) * | 2021-12-04 | 2024-04-05 | 辽宁成大生物股份有限公司 | Method for preparing human encephalitis B inactivated vaccine and vaccine |
| CN114209823A (en) * | 2022-01-23 | 2022-03-22 | 中牧实业股份有限公司 | Adjuvant for pig Japanese encephalitis live vaccine composition, composition and application |
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| CN114317414A (en) * | 2022-01-23 | 2022-04-12 | 中牧实业股份有限公司 | Full suspension cell strain of vero cell and application thereof |
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