CN105833266A - Adjuvant capable of improving immune effect of fish soaking vaccine and fish soaking immunization method - Google Patents
Adjuvant capable of improving immune effect of fish soaking vaccine and fish soaking immunization method Download PDFInfo
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- CN105833266A CN105833266A CN201610263394.9A CN201610263394A CN105833266A CN 105833266 A CN105833266 A CN 105833266A CN 201610263394 A CN201610263394 A CN 201610263394A CN 105833266 A CN105833266 A CN 105833266A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55577—Saponins; Quil A; QS21; ISCOMS
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
- A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2720/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsRNA viruses
- C12N2720/00011—Details
- C12N2720/12011—Reoviridae
- C12N2720/12034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
Abstract
The invention belongs to the field of immunization, prevention and treatment of aquatic organism diseases, and particularly relates to an adjuvant capable of improving the immune effect of a fish soaking vaccine and a fish soaking immunization method. The adjuvant comprises the following components in parts by weight: azone, saponin, glycollic acid, glycerinum and glucoside. The invention further provides the fish soaking immunization method which comprises the following steps: (A) mixing the adjuvant and water to obtain an adjuvant solution, and taking one part of adjuvant solution to mix with a vaccine solution evenly to obtain a soaking immune solution; (B) putting to-be-immunized fish into the adjuvant solution to soak, and then fishing up and cleaning the fish with water; and (C) transferring the treated to-be-immunized fish into the soaking immune solution, and aerating and soaking the to-be-immunized fish. By the adjuvant, the quantity of the vaccine entering the fish body in the soaking immunization process can be effectively improved; the immune effect on a live vaccine (GCHV-892 plant) of a hemorrhagic disease of grass carp is significantly improved; and the adjuvant has the advantages of being convenient to operate, low in cost, safe to fish and the like.
Description
Technical field
The invention belongs to aquatile disease immune prevention and control field, particularly relate to a kind of raising fishing soaking vaccine
The adjuvant of immune effect and fish immersion immunization method.
Background technology
Grass carp is one of important cultured freshwater fish of China, accounts for critical role in China's freshwater aquiculture.Grass
Fish meal source is wide, fast growth, and delicious meat is liked by numerous culturists and consumer deeply.But,
Grass carp disease is many, cultivation survival rate is low, hampers the development of grass carp aquaculture.Hemorrhagic disease of grass carp is a kind of tight
Heavily endangering the viral disease of Grass Carp Juveniles, its Epidemic Scope is wide, spread speed fast, and the death rate is high, every year
All grass carp aquaculture is caused huge economic loss.For a long time, the Study of Prevention Technology of hemorrhagic disease of grass carp
The always important content of China's aquiculture disease Study of Prevention Technology.
The cardinal symptom of hemorrhagic disease of grass carp is around upper lower jaw, oral cavity, eye socket, fin ray base portion, the gill and the gill cover
Hyperemia, exophthalmos '；Peel off the skin of fish, can be seen that muscle has point-like or block congested, hemorrhage, even
Whole-body muscle is cerise；Intestinal wall is congested or hemorrhage, enteral without food, enteron aisle peripheral adipose, mesenterium,
Gall-bladder, the gill, spleen, kidney, liver also have blutpunkte or trace of blood symptom.The symptom of this disease is divided into three types: red
Fin red gill cover type, red muscularity and enteritis type.
Grass carp hemorrhage virus is the infective pathogen of viral hemorrhagic disease of grass carp, is the first fish of China's separation
Virus, is subordinate to Reoviridae, and Aquareovirus belongs to, diameter 70～80nm, icosahedron
Spheric granules, containing the double-stranded RNA of 11 fragments.There is different strains in different regions.Report at present
10, road separates strains, and this virus mainly causes CHINESE FRESHWATER cultivation principal item grass carp to occur in fingerling stage
Hemorrhage, because it colonizes in Fish tissue cell, has the strongest resistance to the action of a drug, so difficulty drug therapy.
At present, the method for the most commonly used vaccine immunity of prevention method of hemorrhagic disease of grass carp, including injection epidemic disease
Seedling, soaking vaccine and oral vaccine etc..Domestic fish vaccine product formulation kind is few, in vaccines for fish
Research aspect lays particular emphasis on injection vaccine more.Vaccinate hemorrhagic disease of grass carp prevention effect obvious, but injection
Immunization amount is big, inefficiency, and can damage fish body；And during immersion immunity, anti-
The former obstacle needing to overcome fish skin mucus, scale and skin, could enter body, and immune effect is poor.
Based on above-mentioned present situation, it is provided that novel immersion vaccine adjuvant, improve vaccines for fish immersion process further,
Improve vaccines for fish immune effect, be necessary to grass carp aquaculture fast development.
Summary of the invention
The present invention is in order to make up the deficiencies in the prior art, it is provided that a kind of raising fishing soaking vaccine immune effect
Adjuvant and fish immersion immunization method.It enters the amount of vaccine of fish body during being effectively improved immersion immunity,
Thus significantly improve the immune effect of grass carp hemorrhage disease live-vaccine (GCHV-892 strain), and its low cost,
To fish safety.
To achieve these goals, technical scheme is as follows: a kind of raising fishing soaking vaccine immunity
The adjuvant of effect, described adjuvant includes the component of following parts by weight: azone 0.01-1 part, saponarin 2-5
Part, glycolic 3.5-6 part, glycerine 10-20 part, glucoside 0.5-3 part.
Preferably, described adjuvant includes the component of following parts by weight: azone 0.01 part, saponarin 2 parts,
Glycolic 3.5 parts, glycerine 10 parts, glucoside 0.5 part.
Preferably, described adjuvant includes the component of following parts by weight: azone 0.05 part, saponarin 3 parts,
Glycolic 5 parts, glycerine 15 parts, glucoside 2 parts.
Preferably, described adjuvant includes the component of following parts by weight: azone 1 part, saponarin 5 parts, second
Alkyd 6 parts, glycerine 20 parts, glucoside 3 parts.
Preferably, described adjuvant also includes shea butter, shea butter and the weight ratio of azone in described adjuvant
It is 1: 0.1-0.6.
Preferably, in described adjuvant, the weight ratio of shea butter and azone is 1: 0.3.
Present invention also offers a kind of fish immersion immunization method, comprise the following steps:
A) above-mentioned adjuvant and deionized water are mixed to get assist agent solution by the volume ratio of 1: 40-60, standby；
B) a part of step A is taken) gained assist agent solution mixes according to the volume ratio of 1: 120-200 with vaccine solution
Uniformly, obtain immersion immunity solution, standby；
C) first will put into step A until immunity fish) assist agent solution of gained soaks 10-30min after pick up, use
Water cleans 1-2min；
D) will be through above-mentioned steps C) process after treat that immunity fish move into step B) the immersion immunity solution of gained
In, 30-60min is soaked in inflation, i.e. completes immunity.
Preferably, described step A) in the volume ratio of adjuvant and deionized water be 1: 45-55.
Preferably, described step B) described in the volume ratio of assist agent solution and vaccine solution be 1: 150-200.
Preferably, described step B) described in the volume ratio of assist agent solution and vaccine solution be 1: 160.
Compared with prior art, the present invention has a following technical advantage:
Fishing soaking vaccine adjuvant of the present invention enters the amount of vaccine of fish body during being effectively improved immersion immunity,
Thus significantly improve the grass carp hemorrhage disease live-vaccine (GCHV-892 strain) immunoprotection to grass carp, its immunity
Method is simple, low cost, to fish safety, is conducive to large-scale production.
Detailed description of the invention
It will be understood by those skilled in the art that what technology representative disclosed in following example the inventors discovered that
Play the technology of good action in the practice of the invention.But, can in disclosed specific embodiments
To make many changes, and still obtain same or analogous result, without deviating from spirit and the model of the present invention
Enclose.
Embodiment 1: a kind of adjuvant improving fishing soaking vaccine immune effect
Described adjuvant is made up of the component of following parts by weight: azone 0.01 part, saponarin 2 parts, glycolic
3.5 parts, glycerine 10 parts, glucoside 0.5 part.
Embodiment 2: a kind of adjuvant improving fishing soaking vaccine immune effect
Described adjuvant is made up of the component of following parts by weight: azone 0.05 part, saponarin 3 parts, glycolic
5 parts, glycerine 15 parts, glucoside 2 parts.
Embodiment 3: a kind of adjuvant improving fishing soaking vaccine immune effect
Described adjuvant is made up of the component of following parts by weight: azone 1 part, saponarin 5 parts, glycolic 6
Part, glycerine 20 parts, glucoside 3 parts and shea butter 0.3 part.
Embodiment 4: a kind of adjuvant improving fishing soaking vaccine immune effect
Described adjuvant is made up of the component of following parts by weight: azone 0.08 part, saponarin 4 parts, glycolic
4.5 parts, glycerine 12 parts, glucoside 1.5 parts and shea butter 0.0048 part.
Fish immersion immunization method
A) above-mentioned adjuvant and deionized water are mixed to get assist agent solution by the volume ratio of 1: 50, standby；
B) a part of step A is taken) gained assist agent solution mixes all according to the volume ratio of 1: 160 with vaccine solution
Even, obtain immersion immunity solution, standby；
C) step A will be put into until immunity fish) assist agent solution of gained soaks 60min after pick up, clear with water
Wash 1min；
D) will be through above-mentioned steps C) process after treat that immunity fish move into step B) the immersion immunity solution of gained
In, 20min is soaked in inflation, i.e. completes immunity.
Comparative example 1: a kind of adjuvant improving fishing soaking vaccine immune effect
Glycolic increases to 6.5 parts, other compositions and parts by weight thereof such as embodiment 3.
Comparative example 2: a kind of adjuvant improving fishing soaking vaccine immune effect
The parts by weight of shea butter increase to 1 part, and other components and parts by weight are same as in Example 3.
Comparative example 3: fish immersion immunization method
S1: take adjuvant and mix to obtain assist agent solution with deionized water by the volume ratio of 1: 50, standby；
S2: take step S1 gained assist agent solution and mix to obtain immersion immunity with vaccine solution by the volume ratio of 1: 160
Solution, standby；
S3: will treat that immunity fish are put into inflation in the immersion immunity solution of step S2 gained and soak 60min, i.e.
Complete immunity.
Comparative example 4: fish immersion immunization method
S1: take adjuvant and mix to obtain assist agent solution with deionized water by the volume ratio of 1: 50, standby；
S2: by picking up after immunity fish are put into and soak 20min in the assist agent solution of step S1 gained, use water
Clean 1min；
S4: treating after above-mentioned steps S2 processes, in immunity fish immigration vaccine solution, is inflated and soaked
60min, i.e. completes immunity.
Toxicity test
(1) test fish: 8-10g grass carp seedling 40 tails/group, totally 6 groups.
(2) test packet:
Embodiment of the present invention 1-4 and comparative example 1-2 group gained adjuvant are mixed according to the volume ratio of 1: 50 with water
Close uniform assist agent solution, be respectively designated as embodiment 1 group, embodiment 2 groups, embodiment 3 groups, enforcement
Example 4 groups, comparative example 1 group and 2 groups of assist agent solutions of comparative example.
(3) processing method: take 6 groups of grass carp seedlings and move into embodiment 1-4 group and comparative example 1-2 group respectively
Soaking half an hour in assist agent solution, observed and recorded respectively organizes grass carp seedling state after soaking 7 days and death condition.
(4) result of the test.
Result of the test: each group grass carp seedling abnormal state does not occurs in 7 days after steeping, death does not occurs yet
Situation.
The impact of hemorrhagic disease of grass carp vaccine immunity protected effect is tested by different immersion immunity methods
1. material:
1.1 test fishes: 8-10g grass carp seedling 450 tail, support 2 weeks temporarily before test, observe feeding activity state,
It is used for testing after confirming health, water temperature (26 ± 1) DEG C during test.
1.2 vaccine solution and strong malicious preparation: GCHV GCHV inoculation grass carp nephrocyte (CIK),
Cultivate 6-8 days for 28 DEG C, collect cell and i.e. obtain vaccine solution with supernatant.Strong poison uses healthy grass carp to prepare,
Healthy grass carp infectable infection contains the tissue fluid of GCHV, and after 4-7 days, symptom occurs in fish body, collects
Suffer from the sick fish tissues of typical case's hemorrhagic disease of grass carp, take the grinding of the viscera tissue such as disease fish spleen, kidney, tissue fluid by centrifugation,
Obtain poison by force after filtration sterilization, test for Immunization.
2 test packets
Blank group: test fish processes without any surface and directly attacks poison；
Test 1. group: adjuvant described in Example 3 according to immersion immunity method described in comparative example 3 to test fish
Carry out immersion immunity；
Test 2. group: adjuvant described in Example 3 according to immersion immunity method described in comparative example 4 to test fish
Carry out immersion immunity；
Test 3. group: test fish is carried out by the immersion immunity method that Example 3 adjuvant provides according to the present invention
Immersion immunity.
Test 4. group: fish processes without any surface in test, directly soak 20min by the vaccine solution of 1.2 preparations
Immunity.
Below often group sets 3 parallel group, each parallel group of 30 tail grass carp seedlings.
Poison is attacked: immunity is respectively organized and attacked with the strong venom of GCHV of 1.2 preparations for latter 15 days after 2.1 immunity
Poison, attacks poison and uses infectable infection mode.
2.2 result of the tests: after attacking malicious 15 days, add up each group of survival rate, the results are shown in Table 1.
Table 1 immunity relative protection ratio
Note: relative immunity protective rate computational methods: RPS=(the 1-test group death rate/control group death rate) ×
100%
As seen from the above table, each group average relative protective rate is ordered as from high to low: test 3. group ＞ tests 1. group
＞ test 2. group ＞ tests 4. group, and wherein, the average relative protective rate that 3. test is organized is 64.8%, higher than examination
Test 50.0% 1. organized and test 46.5% 2. organized.Result of the test shows, utilizes the adjuvant of the present embodiment 3
After pre-processing fish body, then the immersion immunity solution mixed via adjuvant and vaccine solution soaks,
The soaking vaccine immanoprotection action to fish body can be significantly improved.
The adjuvant of the present invention impact on immune effect of vaccine
1. material
1.1 test fishes: 8-10g grass carp seedling 210 tail, support 2 weeks temporarily before test, observe feeding activity state,
It is used for testing after confirming health, water temperature (26 ± 1) DEG C during test.
1.2 Strain: GCHV GCHV-892 strain, limited purchased from the big China in Zhaoqing agriculture biologics
Company.
1.3 adjuvants: embodiment of the present invention 1-4 and comparative example 1-2 gained adjuvant.
The preparation of 1.4 vaccine solution: grass carp kiss end fibroblast (PSF) is passed on according to a conventional method, passes on
Rear cell confluency degree reaches inoculation GCHV-892 Strain during 80%-90%, cultivates 5-6 days for 28 DEG C, waits to turn
Bottle inner cell pathology is complete, collects cell liquid and carries out steriling test and mycoplasma inspection, and it is sick to sample detection
Poison content (TCID50), viral level (TCID50) equal to or more than 1 × 108Time, keep sample standby.
2 test methods
Above-mentioned 210 tail grass carp seedlings are equally divided into 7 groups, often organize 30 tails.Take embodiment of the present invention 1-4 and
Comparative example 1-2 gained adjuvant the most first mixes to obtain embodiment 1-4 and comparative example with water by the volume ratio of 1: 50
1-2 group assist agent solution, take an above-mentioned part respectively organize assist agent solution respectively with vaccine solution according to 1: 160 volume
Ratio mixes to obtain embodiment 1-4 and comparative example 1-2 group immersion immunity solution, marks, standby.Take it
In 6 groups of grass carp seedlings be first respectively put in embodiment of the present invention 1-4 and comparative example 1-2 group assist agent solution immersion
Pick up after 20min, clean 1min with water, then 6 groups of grass carp seedling correspondences respectively after above-mentioned process are put
Enter inflation in above-mentioned each group of immersion immunity solution to soak；Take remaining 1 group of grass carp seedling and first put into leaching in breeding water
Bubble 20min, rear immigration in vaccine solution is soaked as a control group.
2.3 sampling
Respectively to respectively in time soaking 10min, 20min, 40min, 50min, 60min, 1.5h, 2h, 3h
Group soaks fry sampling, and each sample point takes the kidney of 3-5 tail fish and mixes is a sample, and sample collection is at RNA
Preserving in liquid, after sample all takes, the relative amount of vaccine in mensuration fish body, records and analyzes.
2.4 result of the tests such as table 2.
The relative amount of grass carp hemorrhage disease live-vaccine in fish body after the process of table 2 different condition
Note: the computational methods of vaccine relative amount: with the specific gene VP6 of GCHV in fish body kidney
Expression be testing index, use Roche Light cycler480 PCR instrument to measure VP6 base in each sample
The expression of cause.Specifically comprise the following steps that and the sample being collected is extracted respectively RNA, anti-with kit immediately
It is transcribed into cDNA, design VP6 gene and the real-time quantitative PCR primer of grass carp 18S gene, 18S conduct
Reference gene.TOYOBO company real-time quantitative PCR kit is used to be analyzed, every hole PCR system
As follows: Mix 5 μ L, upstream primer 0.4 μ L, downstream primer 0.4 μ L, template cDNA 1 μ L, add sterilizing
Water is to 10 μ L, and each sample does four repetitions.
Result of the test shows: in embodiment 1-4 group fish body, the relative amount of vaccine is in the inspection of different time points
Survey result is all apparently higher than comparative example 1-2 group, especially the most optimum with 3 groups of effects of embodiment, therefore embodiment 3
Group is preferred embodiment.
Owing to describing the present invention by above preferred embodiment, in the spirit and/or scope of the present invention,
The present invention is implemented in any replacement/for the present invention or combination, is the most all
It will be apparent that and be included among the present invention.
Claims (10)
1. the adjuvant improving fishing soaking vaccine immune effect, it is characterised in that described adjuvant include with
The component of lower parts by weight: azone 0.01-1 part, saponarin 2-5 part, glycolic 3.5-6 part, glycerine 10-20
Part, glucoside 0.5-3 part.
2. the raising fishing adjuvant of soaking vaccine immune effect as claimed in claim 1, it is characterised in that
Described adjuvant includes the component of following parts by weight: azone 0.01 part, saponarin 2 parts, glycolic 3.5 parts,
Glycerine 10 parts, glucoside 0.5 part.
3. the raising fishing adjuvant of soaking vaccine immune effect as claimed in claim 1, it is characterised in that
Described adjuvant includes the component of following parts by weight: azone 0.05 part, saponarin 3 parts, glycolic 5 parts,
Glycerine 15 parts, glucoside 2 parts.
4. the raising fishing adjuvant of soaking vaccine immune effect as claimed in claim 1, it is characterised in that
Described adjuvant includes the component of following parts by weight: azone 1 part, saponarin 5 parts, glycolic 6 parts, sweet
Oil 20 parts, glucoside 3 parts.
5. the raising fishing adjuvant of soaking vaccine immune effect as claimed in claim 1, it is characterised in that
Described adjuvant also includes shea butter, and in described adjuvant, the weight ratio of shea butter and azone is 1: 0.1-0.6.
6. the raising fishing adjuvant of soaking vaccine immune effect as claimed in claim 5, it is characterised in that
In described adjuvant, the weight ratio of shea butter and azone is 1: 0.3.
7. a fish immersion immunization method, it is characterised in that comprise the following steps:
A) taking adjuvant described in any one of claim 1-6 and water, to be mixed to get adjuvant by the volume ratio of 1: 40-60 molten
Liquid, standby；
B) a part of step A is taken) gained assist agent solution mixes according to the volume ratio of 1: 120-200 with vaccine solution
Uniformly, obtain immersion immunity solution, standby；
C) first will put into step A until immunity fish) assist agent solution of gained soaks 10-30min after pick up, use
Water cleans 1-2min；
D) will be through above-mentioned steps C) process after treat that immunity fish move into step B) the immersion immunity solution of gained
In, 30-60min is soaked in inflation, i.e. completes immunity.
8. fish immersion immunization method as claimed in claim 7, it is characterised in that described step A) in
Adjuvant is 1: 45-55 with the volume ratio of water.
9. fish immersion immunization method as claimed in claim 7, it is characterised in that described step B) in
Described assist agent solution is 1: 150-200 with the volume ratio of vaccine solution.
10. fish immersion immunization method as claimed in claim 9, it is characterised in that described step B)
Described in the volume ratio of assist agent solution and vaccine solution be 1: 160.
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| CN201610263394.9A CN105833266A (en) | 2016-04-26 | 2016-04-26 | Adjuvant capable of improving immune effect of fish soaking vaccine and fish soaking immunization method |
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| CN108524927A (en) * | 2018-05-17 | 2018-09-14 | 广州普麟生物制品有限公司 | A kind of grass carp hemorrhage disease inactivated vaccine and preparation method thereof |
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| CN108524927A (en) * | 2018-05-17 | 2018-09-14 | 广州普麟生物制品有限公司 | A kind of grass carp hemorrhage disease inactivated vaccine and preparation method thereof |
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