CN103599532A - Chicken infectious bronchitis divalent live vaccine and preparation method thereof - Google Patents
Chicken infectious bronchitis divalent live vaccine and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a chicken infectious bronchitis divalent live vaccine and a preparation method thereof. The divalent live vaccine comprises antigens and a freeze-drying protective agent. The antigens comprise a chicken kidney-type infectious bronchitis virus K136 strain having an accession number of CCTCC-V201320 and a chicken breath-type infectious bronchitis virus H120 strain. The preparation method includes respectively performing amplification culture to the IBV-K136 strain and the H120 strain through chick embryos; mixing culturing products uniformly to prepare the vaccine antigens; and mixing uniformly the vaccine antigens and the freeze-drying protective agent and performing freeze drying to prepare the vaccine. The divalent live vaccine can prevent the chicken breath-type infectious bronchitis and the chicken kidney-type infectious bronchitis simultaneously. The vaccine conforms to the epidemiological characteristics of the chicken infectious bronchitis in our country at present, and solves a problem that no vaccine for the chicken kidney-type epidemic infectious bronchitis is available in our country at present. The divalent live vaccine can be popularized and used in a large scale.
Description
Technical field
The invention belongs to biological new medicine technical field for poultry, especially relate to a kind of infectious bronchitis of chicken bivalent vaccine and preparation method thereof.
Background technology
Infectious bronchitis of chicken (Avian Infectious Bronchitis, IB) is a kind of acute, the height contagious disease of chicken, and it mainly encroaches on respiratory system, the genito-urinary system digestive system of unifying.Primary disease is reported by U.S. Schalk and Hawn the earliest, clinical take suffer from that pertussis is coughed, the respiratory symptom of sneeze and trachea rale is as main, in countries in the world, occur successively afterwards, Beach in 1936 and Schalm determine that cause of disease is virus (breathing pattern IBV), rear called after avian infectious bronchitis virus (Infectious Bronchitis Virus, IBV), being included into coronaviridae, is the representative strains of coronavirus.Kuang Rong Lu in 1972 etc. confirm that China also has this disease to occur first in Guangdong.Infectious bronchitis virus serotype is more, common are Massachussetts, Connecticat, lowa97, Iowa609, Hotle, JMK, Clark333, SEl7, Florida, Arkanass99 and Australian " T ".The reported first such as Wintefield in 1962 kidney type IBV, mainly cause kidney enlargement, urate deposition, outward appearance presents typically " piebaldism kidney ".
Infectious bronchitis of chicken is worldwide distribution, very harmful to poultry husbandry, weightening finish and the price of deed that it can make into chicken reduce, the Yield and quality of egg and incubation rate decline, and directly cause that chickling is dead, nephropathy and the permanent degeneration of fallopian tube; In addition, IB also often participates in mixed infection as one of cause of disease, for example, bring out the outburst of chronic respiratory tract disease (CRD).Due to the discontinuous mechanism of IBV genomic nucleic acids rna replicon and the incomplete check and correction mechanism of RNA polymerase, make viral RNA easily producer point mutation, insertion, disappearance or restructuring in reproduction process, therefore cause the appearance of a large amount of IBV variants.The IB serotype being separated in the world has at present reached 30 kinds more than, only has part or without cross-protection between different serotypes strain vaccine, and many serotype of IB has increased difficulty for its immunoprophylaxis.
In recent years, in world wide, infectious bronchitis of chicken is popular in rising trend, and especially nephropathy modification IB has spread to dozens of countries and regions, has caused serious economic loss to local poultry husbandry.According to Epidemiological study result, show, IBV is popular in most of China area at present, and exists with multiple pathological form simultaneously, mainly take by " kidney type " and " breathing pattern " virus as main.Present stage, China mainly comprised H120 strain, M41 strain etc. for the preparation of the weak poison of vaccine control breathing pattern IB, and the weak poison of classical kidney type mainly comprises W93 strain, Ma5 strain etc.Variation along with IBV virus, vaccine prepared by most of classical breathing pattern low virulent strain is eliminated by market gradually, and vaccine prepared by H120 strain is pursued by market owing to meeting the current popular breathing pattern IB of China, with the classical strain of most of breathing pattern IB be similarly the preventing efficiency that the at present popular kidney type IB of Liao Dui China is also lost in W93 strain gradually, and the vaccine of preparing with this strain is because its side reaction is larger, be not suitable for the control to the epidemic strain of China, therefore screening obtains kidney type IB strain that new protection is good and for the preparation of just seeming particularly important for the popular renal type infectious bronchitis viral lived vaccine of current China.
China does not still have to prevent and treat the commercialization bivalent vaccine of Testis et penis Gallus domesticus type and respiratory infectious bronchitis simultaneously at present; for effectively preventing and treating " kidney type " and " breathing pattern " virus wantonly plunderring China's poultry husbandry; often need to be with corresponding univalent vaccine to cultivation chicken group immunity repeatedly; strengthened being exempted from chicken group's immunological stress; strengthened greatly the consumption of human and material resources costs simultaneously; the existing commercially available vaccine of what is more important does not often have protective effect to kidney type infectious bronchitis of chicken, often causes immuning failure.
Summary of the invention
The object of the invention is to be to provide a kind of infectious bronchitis of chicken bivalent vaccine, the effect that realization can effectively prevent breathing pattern and kidney type infectious bronchitis of chicken simultaneously, solve that domestic existing IBV vaccine at present can only provide partial prophylaxis to above-mentioned two types of infectious bronchitis of chickens or even without the problem of any preventive effect, thereby avoid to a greater extent the extensive generation of infectious bronchitis of chicken and popular.
The present invention also aims to provide the preparation method of above-mentioned a kind of infectious bronchitis of chicken bivalent vaccine; be about to the weak malicious K136 strain of kidney type infectious bronchitis of chicken and the weak malicious H120 strain of breathing pattern infectious bronchitis of chicken respectively after Embryo Gallus domesticus amplification culture; the cultured products geometric ratio of results is mixed and made into vaccine antigen, then after vaccine antigen is mixed with freeze drying protectant equal-volume, vaccine product is made in lyophilizing.
For realizing above-mentioned purpose of the present invention, the technical scheme adopting is as follows:
An infectious bronchitis of chicken bivalent vaccine, this live vaccine is comprised of antigen and freeze drying protectant, and described antigen comprises Nephropathogenic infectious bronchitis virus and chicken respiratory infectious bronchitis virus.Described freeze drying protectant comprises 10% sucrose and 5% skimmed milk.
In above-mentioned bivalent vaccine, the Nephropathogenic infectious bronchitis virus that described antigen comprises refers to that deposit number is the K136 strain of CCTCC-V201320; The chicken respiratory infectious bronchitis virus that described antigen comprises refers to H120 strain.
The avian infectious bronchitis virus H120 strain that antigen comprises is all classical vaccine antigens, and the vaccine of preparing with it has good prevention effect to the popular newcastle of China and chicken respiratory infectious bronchitis.The K136 strain virus that antigen comprises be by inventor for China at present popular Nephropathogenic infectious bronchitis virus in Embryo Gallus domesticus 136 generations of going down to posterity, cause weak continuously.It has effectively overcome take the shortcoming that classical kidney type strain side reaction in vaccine preparation process that W93 strain is representative is large, be not suitable for China's epidemic strain control, does antigen prepare vaccine and still belong to the first time in the present invention with K136 strain.
The present invention also provides a kind of preparation method of infectious bronchitis of chicken bivalent vaccine; described preparation method is characterised in that it is to select avian infectious bronchitis virus K136 strain and H120 strain also respectively after Embryo Gallus domesticus amplification culture; cultured products mix homogeneously is prepared into vaccine antigen, then lyophilizing after vaccine antigen and freeze drying protectant mix homogeneously is made to vaccine.
A preparation method for above-mentioned bivalent vaccine, comprising following steps:
1) select avian infectious bronchitis virus K136 strain and H120 strain;
2) respectively by IBV-K136 strain and IBV-H120 strain through allantoic cavity inoculated into chick embryo, carry out viral amplification culture;
3) gather in the crops respectively above-mentioned two-strain scale-up medium, through aseptic process and carry out after viral level mensuration the antigen of equal-volume mix homogeneously preparation cost invention vaccine;
4) vaccine antigen is mixed homogeneously with freeze drying protectant equal-volume after lyophilizing, the vaccine in preparation cost invention.
Above-mentioned preparation method specifically comprises the following steps:
1) select avian infectious bronchitis virus K136 strain and the H120 strain of preservation, for viral amplification culture;
2) respectively malicious H120 strain a little less than malicious K136 strain and breathing pattern infectious bronchitis of chicken a little less than kidney type infectious bronchitis of chicken is inoculated in to 10 age in days SPF Embryo Gallus domesticus through allantoic cavity; Hatch after 50-60 hour for 37 ℃ and gather in the crops respectively whole chick embryo allantoic liquids as viral scale-up medium;
3) above-mentioned cultured products carried out respectively to aseptic process and measured after viral level, two-strain scale-up medium product geometric ratio is mixed as semi-finished product, being the antigen of vaccine in the present invention;
4) vaccine antigen and freeze drying protectant are carried out to proportioning according to volume ratio 1:1, after lyophilization, after 36-48h, obtain vaccine, be finished product.
Bivalence Seedling prepared by the present invention can prevent breathing pattern and kidney type infectious bronchitis of chicken simultaneously, meets the epidemiological features of the current infectious bronchitis of chicken of China, can promote on a large scale and use.And the present invention's current epidemic isolates of K136 Zhu Wei China of screening acquisition low virulent strain that repeatedly goes down to posterity, its product has been filled up China's blank without Miao Keyong for popular kidney type infectious bronchitis of chicken at present, itself and classical breathing pattern infectious bronchitis of chicken H120 strain are made to bivalent vaccine simultaneously, can effectively reduce the immunological stress of being exempted from chicken group, greatly reduce the consumption of human and material resources cost simultaneously.
The specific embodiment
For further illustrating content of the present invention, Characteristic, hereby exemplify following examples and further describe the present invention.It should be appreciated by those skilled in the art, under prerequisite without departing from the spirit and scope of the present invention, can the details of technical solution of the present invention and form be modified or be replaced, but these modifications and replacement all fall into protection scope of the present invention.
Embodiment 1: the screening of infectious bronchitis of chicken kidney type strain K136 strain
Our company gathered doubtful avian infectious bronchitis virus isolated strain at the different chicken houses of China respectively from 2003-2005.Sick chicken main manifestations is lassitude, cough, often hear snore, pathology cuts open inspection and finds that degasification pipe ring has kidney outside little petechia to also have obvious pathological changes, through Preliminary Identification, obtain 4 strain renal type infectious bronchitis virus stains, called after CK/IBV/QD03 strain respectively, CK/IBV/ZC03 strain, CK/IBV/YT04 strain and CK/IBV/SD05 strain.A little less than using 10 age in days SPF Embryo Gallus domesticus that four strain strains are carried out continuous passage and caused, in per generation, is inoculated 4 pieces of Embryo Gallus domesticus by allantoic cavity, hatch for 37 ℃, and the 36h Embryo Gallus domesticus of freezing to death after inoculation, results allantoic fluid, for inoculation of future generation, connects and reached for 136 generations.Cause during this time weak evaluation, its evaluation methodology is to get the virus strain infection in the 2nd, 20,40,50,60,70,80,90,100,110,120,124,128,132,134,136,140 generations 1 Japanese instar chickling, observes chickling incidence.
The results are shown in Table 1:
The pathogenic result of the different generations of 4 kinds of separated strains of table 1 to SPF chicken
Table 1 is continuous
Result shows to only have CK/IBV/SD05 strain to start without dead from reaching the tested chicken of the 80th generation Strain, and the tested chicken of the 100th generation Strain starts without observing pathological changes, the 120th generation Strain start tested chicken and start inorganization and learn pathological changes.Other 3 strains its viral titer a little less than not high this shows that virus can not well adapt to, cause on Embryo Gallus domesticus always in the process of going down to posterity; M & M is not all 0, a little less than proving and not causing after these three kinds of strains go down to posterity on Embryo Gallus domesticus, the in the situation that even some strain being a little less than causing, occurred again returning by force, these all illustrate the vaccine strain use that low virulent strain is all not suitable as live vaccine that causes of CK/IBV/QD03 strain, CK/IBV/ZC03 strain and CK/IBV/YT04 strain.
And a little less than CK/IBV/SD05 strain causes completely in the 120th generation, tested chicken is had to good safety.Simultaneously with the immune 3 age in days SPF chickens of the 124th, 128,132,134,136 generation poison difference; carry out immunoprotection evaluation; homology strong virus attack is used in immunity for latter 14 days; immune component poison rate is 0/10; contrast component poison rate is 10/10; illustrate that strain of the present invention has good immune protective; for ease of distinguishing with former strain; the CK/IBV/SD05 strain that is passaged to the reduction of 136 generations is decided to be to original species poison E1 generation; the called after coronavirus genus avian infectious bronchitis virus avian infectious bronchitis virus K136 of section strain, candidate's strain of preparing as vaccine in the present invention.Avian infectious bronchitis virus K136 of the present invention strain is preserved in Wuhan University's Chinese Typical Representative culture collection center on June 28th, 2013, and deposit number is: CCTCC-V201320.
Embodiment 2:K136 strain safety detects
1, the malicious valency of different generation K136 strains on Embryo Gallus domesticus measured
Get E2, E4, E7, E10, the E15 of K136 strain for seed culture of viruses, do suitable multiple dilution, through allantoic cavity, inoculate 10 age in days SPF Embryo Gallus domesticus, every embryo 0.1mL arranges blank group simultaneously, and every embryo, through allantoic cavity inoculation normal saline 0.1mL, is hatched cultivation for 37 ℃.Discard dead Embryo Gallus domesticus in 24h, hatch after 144h, take out the chick embryo development situation of observing.According to Reed-Muench method, calculate EID
50, E2, E4, E7, E10, E15 are respectively for the malicious valency of seed culture of viruses: 10
6.7, 10
6.8, 10
6.5, 10
6.6, 10
6.8eID
50/ 0.1mL.Result of the test explanation K136 strain is bred stable on Embryo Gallus domesticus, and avirulence is returned strong phenomenon.
2, the pathogenicity of different generation K136 strains on chickling
Choose 10 age in days SPF chickling, be divided at random 6 groups, every group of 10 chickens, respectively with 10
5.0eID
50collunarium inoculation is got E2, E4, E7, E10, the E15 of K136 strain for seed culture of viruses 0.1mL, last group is usingd normal saline collunarium 0.1mL as blank group, each organizes all separately isolated rearings of tested chicken, after Continuous Observation 20 days, slaughter, cut open inspection and respectively organize tested chicken and whether occur the characteristics of lesion such as tracheorrhagia, secretions increase, renomegaly, urate deposition, the results are shown in Table 2.
The safety testing result of the different generation K136 strains of table 2 on chickling
Above-mentioned result of the test shows, respectively organizes all strong living of tested chicken after off-test, without any abnormal response, cuts open inspection inorganization and learns pathological changes, and this shows that K136 strain virulence on chickling is stablized and virulence does not occur returns by force.
Embodiment 3: different generation K136 strain immune efficacies are evaluated
With E2, E4, E7, E10, E15 generation K136 strain seed culture of viruses chick embryo allantoic liquid, with 5 * 10
2.0eID
50dosage, to 1 age in days SPF chicken collunarium, latter 14 days of immunity, together with contrast chicken, every chicken is with 10
3.68eID
50the strong malicious CK/IBV/SD05 strain eye dripping of separated strain of/0.1mL, each 0.05mL of collunarium, after counteracting toxic substances the 5th day, gather the trachea swab of every chicken, through allantoic cavity, inoculate 5 pieces of 10 age in days SPF Embryo Gallus domesticus, 0.1mL/ embryo, hatches and observes to 144 hours.Result demonstration, immune component poison rate is between 0/10-1/10, and protective rate is between 9/10-10/10; Contrast component poison rate is 10/10.Illustrate that IBV-K136 strain seed culture of viruses is basically identical with interior its immunogenicity in 15 generations, all there is good immunogenicity, illustrate that IBV-K136 strain has good immune protective.
Embodiment 4: the preparation of infectious bronchitis of chicken bivalent vaccine and check
1. material
Produce with seed culture of viruses avian infectious bronchitis virus H120 strain purchased from China Veterinery Drug Inspection Office; Avian infectious bronchitis virus K136 strain, seed culture of viruses preserving number is CCTCC-V201320.
Check is with malicious avian infectious bronchitis virus M41 strain, purchased from China Veterinery Drug Inspection Office; Separated strain CK/IBV/SD05 Qiang Duyou place company isolation identification.
SPF hatching egg and SPF chicken are purchased from 61 farms, Shandong.
2. produce the breeding with K136 kind poison
Utilize physiological saline solution to carry out 100 times of dilutions the basis kind poison of K136 strain, under aseptic condition, inoculate 10 age in days SPF Embryo Gallus domesticus, each egg inoculation 0.1mL is in allantoic cavity, in the rearmounted 37 ℃ of incubators of paraffin sealing, hatch, discard in 24 hours dead Embryo Gallus domesticus, once every 4-8 hour photograph egg, dead Embryo Gallus domesticus is chosen and is placed in 4 ℃ later, within 36 hours, residue Embryo Gallus domesticus is placed in to 8-12 hour in 4 ℃ of refrigerators, then under aseptic condition, gathers in the crops chick embryo allantoic liquid.Efficacy test, steriling test is qualified be placed on-20 ℃ standby.
3. produce the breeding with H120 kind poison
Utilize physiological saline solution to carry out 100 times of dilutions the basis kind poison of H120 strain, under aseptic condition, inoculate 10 age in days SPF Embryo Gallus domesticus, each chick embryo allantoic cavity inoculation 0.1mL, in the rearmounted 37 ℃ of incubators of paraffin sealing, hatch, discard Embryo Gallus domesticus dead in 24 hours, once every 4-8 hour photograph egg, dead Embryo Gallus domesticus is chosen and is placed in 4 ℃, within 48 hours, gathers in the crops whole chick embryo allantoic liquids later.Efficacy test, steriling test is qualified be placed on-20 ℃ standby.Prepare altogether 3 batches of virus liquids, lot number is respectively 1301,1302,1303.Efficacy test and steriling test the results are shown in Table 3.
Table 3: each batch of seed culture of viruses viral level and steriling test result
4. join Seedling and lyophilizing
K136 strain virus liquid after the assay was approved and H120 strain virus liquid and sucrose skimmed milk protective agent are joined to Seedling in 1:1:2 ratio; the ampicillin and the streptomycin that add 500 units/mL simultaneously; after quantitative separating, carry out lyophilisation process, lyophilizing overall process needs 30-48h.Prepare according to the method described above 3 batch samples, lot number is respectively 1301,1302,1303.
5. product inspection
The outward appearance of 5.1 character observation three batch samples, character, depart from situation and add diluent with bottle wall after dissolving situation.
5.2 steriling tests are tested by < < Chinese veterinary pharmacopoeia > > appendix method.
5.3 mycoplasma checks are tested by < < Chinese veterinary pharmacopoeia > > appendix method.
5.4 diagnostic test
Bivalence Seedling is done to suitable dilution with normal saline, mix with equivalent anti-avian infectious bronchitis virus K136 strain and avian infectious bronchitis virus H120 strain specific antibody, in room temperature, with l hour, allantoic cavity is inoculated 10 pieces of 10 age in days SPF Embryo Gallus domesticus, every embryo 0.2mL.Put 37 ℃ and hatch, 144 hours result of determination.
5.5 safety examination
Safety testing shares 40 10 age in days SPF chickling, and every batch of vaccine is used 10, establishes 10 chickens of matched group simultaneously, every batch of vaccine is selected to 3 bottles at random with the dosage of normal saline dilution to 10 plumage part/0.1mL, test group collunarium inoculation 0.1mL/ only, observes 14, sees whether occur relevant symptoms.
5.6 efficacy test
5.6.1 infectious bronchitis of chicken K136 strain part
With 10 of 3 age in days SPF chickens, the vaccine of every collunarium 1 plumage part.Establish 10 not immune matched groups simultaneously.Latter 14 days of immunity, together with contrast chicken, every chicken collunarium attacks 10
3.68eID
50iBV-K2 strain, observe 10, record incidence.
5.6.2 infectious bronchitis of chicken H120 strain part
With 10 of 3 age in days SPF chickens, the vaccine of every collunarium 1 plumage part.Establish 10 not immune matched groups simultaneously.Latter 14 days of immunity, strong each 0.1mL of malicious collunarium of IBV M41 strain together with every chicken of contrast chicken with 10 times of dilutions, observes 10.Record incidence.
6. product inspection result
6.1 character
3 batches of bivalence Seedling sample appearance are faint yellow Sponge Porosity agglomerate, and while rocking sample, sample easily departs from bottle wall, all dissolve rapidly after adding diluent.
6.2 steriling test
The demonstration of steriling test result, 3 batches of bivalence Seedling samples are all without antibacterial, mould contamination, and sample steriling test is all qualified.
6.3 mycoplasma checks
The demonstration of mycoplasma assay, 3 batches of bivalence Seedling samples are all without mycoplasma growth, and the check of sample mycoplasma is all qualified.
6.4 diagnostic test
3 batches of bivalence Seedlings are done suitable dilution (1 plumage part/mL) with physiological saline solution, mix with the K136 strain of equivalent infectivity resistant bronchitis virus and infectivity resistant bronchitis virus H120 strain specific antibody respectively, in room temperature and l hour, in allantoic cavity, inoculation 10 age in days SPF Embryo Gallus domesticus is 10 pieces, every embryo 0.2mL, put 37 ℃ and hatch, observe 144 hours.Result demonstration, inoculated into chick embryo is 10/10 strong living, and illustrates that bivalence Seedling diagnostic test is qualified.
6.5 safety examination
Every batch of vaccine is selected to 3 bottles at random and with collunarium after normal saline dilution, inoculate 10 age in days SPF chickens, every chicken 0.1mL(is containing 10 plumage parts), observe 14.Result shows, inoculates chicken and all has no adverse reaction, and all strong alive, the results are shown in Table 4.
Table 4 bivalence Seedling safety examination result
6.6 efficacy test
6.6.1 avian infectious bronchitis virus K136 strain part
With 10 of 3 age in days SPF chickens, the vaccine of every collunarium 1 plumage part.Establish 10 not immune comparing simultaneously.Latter 14 days of immunity, together with contrast chicken, every chicken attacks 10 by nasal drip
3.68eID
50iBV-K2 strain observe 10.Record incidence.The results are shown in Table 5.
Table 5 bivalence Seedling efficacy test K136 strain part
6.6.2 avian infectious bronchitis virus H120 strain part
With 10 of 3 age in days SPF chickens, the vaccine of every collunarium 1 plumage part.Establish 10 not immune comparing simultaneously.Latter 14 days of immunity, together with contrast chicken, the strong malicious collunarium 0.1mL of IBV M41 strain of 10 times of dilutions for every chicken, observes 10.Record incidence.The results are shown in Table 6.
Table 6 bivalence Seedling efficacy test H120 strain part
Above experimental result shows, the infectious bronchitis of chicken bivalent vaccine in the present invention can reach simultaneously the effectively effect of prevention breathing pattern and kidney type infectious bronchitis of chicken.
Claims (9)
1. an infectious bronchitis of chicken bivalent vaccine, this live vaccine is comprised of antigen and freeze drying protectant, it is characterized in that described antigen comprise Nephropathogenic infectious bronchitis virus and chicken respiratory infectious bronchitis viral.
2. a kind of infectious bronchitis of chicken bivalent vaccine according to claim 1, is characterized in that the Nephropathogenic infectious bronchitis virus that described antigen comprises refers to that deposit number is the K136 strain of CCTCC-V201320; The chicken respiratory infectious bronchitis virus that described antigen comprises refers to H120 strain.
3. a kind of infectious bronchitis of chicken bivalent vaccine according to claim 1 and 2, is characterized in that K136 strain virus that described antigen comprises is that Embryo Gallus domesticus 136 generations of going down to posterity cause weak acquisition continuously for the at present popular Nephropathogenic infectious bronchitis virus of China by inventor.
4. according to a kind of infectious bronchitis of chicken bivalent vaccine described in claim 1 or 2 or 3, it is characterized in that K136 strain virus that described antigen comprises is in Attenuation is evaluated, reaching the tested chicken of the 80th generation Strain starts without dead, the tested chicken of the 100th generation Strain starts without observing pathological changes, the 120th generation Strain start tested chicken and start inorganization and learn pathological changes.
5. according to a kind of infectious bronchitis of chicken bivalent vaccine described in claim 1 or 2 or 3, it is characterized in that K136 strain virus that described antigen comprises is in immunity evaluation, reaching the malicious rate of the 136th generation Strain immune component is 0/10, and contrast component poison rate is 10/10.
6. the preparation method of an infectious bronchitis of chicken bivalent vaccine; it is characterized in that being to select avian infectious bronchitis virus K136 strain and H120 strain also respectively after Embryo Gallus domesticus amplification culture; cultured products mix homogeneously is prepared into vaccine antigen, then lyophilizing after vaccine antigen and freeze drying protectant mix homogeneously is made to vaccine.
7. the preparation method of a kind of infectious bronchitis of chicken bivalent vaccine according to claim 6, is characterized in that comprising the following steps:
1) select avian infectious bronchitis virus K136 strain and H120 strain;
2) respectively by IBV-K136 strain and IBV-H120 strain through allantoic cavity inoculated into chick embryo, carry out viral amplification culture;
3) gather in the crops respectively above-mentioned two-strain scale-up medium, through aseptic process and carry out after viral level mensuration the antigen of equal-volume mix homogeneously preparation cost invention vaccine;
4) vaccine antigen is mixed homogeneously with freeze drying protectant equal-volume after lyophilizing, the vaccine in preparation cost invention.
8. according to the preparation method of a kind of infectious bronchitis of chicken bivalent vaccine described in claim 6 or 7, the scale-up medium that it is characterized in that two-strain in antigen preparation process is that equal-volume mixes.
9. according to the preparation method of a kind of infectious bronchitis of chicken bivalent vaccine described in claim 6 or 7, it is characterized in that antigen and freeze drying protectant are that equal-volume mixes in vaccine preparation process.
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| CN111707822A (en) * | 2020-08-20 | 2020-09-25 | 兆丰华生物科技(南京)有限公司 | Mycoplasma gallisepticum antibody detection reagent and preparation method and application thereof |
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Application publication date: 20140226 |