CN106085966B - A kind of avian flu strain - Google Patents

A kind of avian flu strain Download PDF

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CN106085966B
CN106085966B CN201610399186.1A CN201610399186A CN106085966B CN 106085966 B CN106085966 B CN 106085966B CN 201610399186 A CN201610399186 A CN 201610399186A CN 106085966 B CN106085966 B CN 106085966B
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avian influenza
vaccine
subtype avian
embryo
influenza virus
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CN106085966A (en
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李陆梅
宋新宇
马爽
宫晓
刘�东
郑欢
郭伟伟
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Qingdao Yebio Bioengineering Co Ltd
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    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine
    • 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
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
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    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

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Abstract

It is an object of the invention to provide one plant of H9 subtype avian influenza strain, deposit number is CCTCC No.V201617.The HA and EID of FJ11 plants of H9 subtype avian influenza virus new strains of the invention50Potency is high, immunogenicity is good and can resist the attack of H9 subtype avian influenza virus each place prevalence separation poison.The vaccine safety that it is prepared is good, does not occur any locally and systemically adverse reaction as caused by vaccine.Analysis in storage life test Jing Guo character, safety testing, potency test data, as a result compared with similar products compared with indices are stable effective.

Description

A kind of avian flu strain
Technical field
The invention belongs to veterinary biologics technical fields, and in particular to a kind of avian flu strain.
Background technique
Bird flu is one of birds deadly infectious disease, and as caused by influenza A, poultry is in infection H9 subtype avian influenza Virus can lead to and cause high mortality and laying rate degradation especially when with mixed infection.It and is Major Epidemic In one of animals and humans infectiousness, the death rate high acute, highly contagious disease, betide extensively all over the world, It is very big to the mankind and aviculture harm.
Variation of the avian influenza virus in birds epidemic disease virus is relatively most frequent, although so that the multiple choices released Vaccine, but still there is losing control of the situation of epidemic situation development, obtain new popular strain so needing to screen and become to cope with New harm caused by different.
Summary of the invention
The purpose of the present invention is filtering out one plant of new H9 subtype avian influenza strain, and its strain is prepared into inactivation epidemic disease Seedling, to make up harm caused by existing strain variation.
Present invention firstly provides a kind of Avian Influenza Virus H9N2 FJ11 plants, China is deposited on March 31st, 2016 Wuhan, Wuhan University China typical culture collection center, deposit number be CCTCC No.V201617.
The present invention also provides a kind of inactivated vaccines, and wherein antigen is the H9 subtype avian influenza virus of inactivation；
Above-mentioned inactivated vaccine, wherein the inactivation of virus uses formalin-inactivated；
Inactivated vaccine of the invention the preparation method is as follows:
1) oil is mutually prepared:
Take 95 parts of mineral oil, 1 part of aluminum stearate, after being uniformly mixed in oily phase preparation tank and being heated to 80 DEG C, then plus 5 parts This 80 (Span-80) of department complete oil after cooling and mutually prepare until temperature maintains 40 minutes when reaching 115 DEG C；
2) prepared by water phase:
95 parts of the H9 subtype avian influenza antigen liquid of inactivation is taken, 5 parts of Tween 80s of sterilizing mix well；
3) it emulsifies
2 parts of oily phase is taken to be put into emulsion tank, after being added 1 part of water phase, then with the 3500r/min stirring cream of completion in 30~40 minutes Change preparation.
The HA and EID of FJ11 plants of H9 subtype avian influenza virus new strains used in vaccine of the invention50Potency is high, immune Originality is good and can resist the attack of H9 subtype avian influenza virus each place prevalence separation poison.The safety of vaccine prepared by the present invention Well, do not occur any locally and systemically adverse reaction as caused by vaccine.It is tried in storage life test by character, safety It tests, the analysis of potency test data, as a result compared with similar products compared with indices are stable effective；Efficacy test results card Bright, it is fast that H9 subtype avian influenza inactivated vaccine antibody generates antibody than similar product, it is important that can resist prevalence after immune The protection of strain.
Specific embodiment
Applicant screens the H9 subtype avian influenza virus for obtaining one plant of novel variant, which is prepared into inactivation epidemic disease Seedling, to facilitate the present invention.
The present invention is described in detail below with reference to embodiment.
The screening of embodiment 1:H9 subtype avian influenza strain
1. epidemiological survey is since the northern area of China in 1998 finds H9 subtype avian influenza, we are just always to H9 The popularity and virus variation situation of subtype avian influenza carry out follow-up study, and in recent years, bird flu occur in a lot of areas The morbidity of symptom or dead chicken, by a large amount of epidemiological investigation, we are successful from Fujian chicken farm within 2015 One plant of H9 subtype avian influenza virus is isolated in chicken group.
2. the separation that the method for virus purification according to the literature makees AIV.Pathological material of disease is added in 1:5 (w/v) ratio and is contained In dual anti-physiological saline, suspension is made in grinding, and 1000r/min is centrifuged 5 minutes, and supernatant allantoic cavity is taken to be inoculated with 10~11 ages in days SPF chicken embryo, every embryo 0.2ml, 36 DEG C~37 DEG C hatch 3~4, every sunshine embryo 2 times, dead chicken embryo after taking 24 hours, set 2~ 8 DEG C cooling 8~16 hours, harvest chicken embryo liquid, carry out HA and HI test.
3.HA and HI test is carried out by existing " Chinese veterinary pharmacopoeia ".
4. viral subculture and viral level measurement the virus subculture in 10~11 age in days SPF chicken embryos, harvest allantoic fluid.Virus Assay uses following method: viral allantoic fluid sterile saline work being serially diluted for 10 times, each dilution is through urinating Inoculation 10~11 age in days SPF chicken embryo each 5, every embryo 0.1ml in blister cavities, is observed 120 hours, calculates EID50。
5. the identification of hypotype inactivates 0.05% formalin of chick embryo allantoic liquid of isolated FJ11 strain and other strains Afterwards, national influenza center is sent, HI and NI test is done, determines the hypotype of virus.
6. FJ11 plants of E0 for allantoic fluid poison, are made 10 with sterile saline by the virulence of pair chicken embryo-4Dilution, inoculation 10~11 Age in days SPF chicken embryo 20, every embryo allantoic cavity inoculation 0.1ml is (containing about 1000EID50), 36~37 DEG C were incubated for 120 hours, discarded Dead chicken embryo before 60 hours, later daily observation twice, record dead embryo number.
7. the virulence of pair chick by " OIE " standard with 4 week old SPF chickens and press " european union directive " standard with 6 week old SPF chickens into Virulence test of the row to chick.
8. Study On Immunogenicity is with 7~10 age in days SPF chickens 20, wherein 10 chicken every inoculate 10 times it is diluted YBF003 plants of allantoic fluids, another 10 subcutaneous vaccinations physiological saline compare, and are individually insulated raising, blood sampling separation serum after 21 days, HI test is made to standard H9 antigen.
9. antigentic specificity is tested
The HI test of 9.1 pairs of H9 subtype specific antiseras by seed culture of viruses respectively with bird flu (H5, H7, H9 hypotype), ND, EDS antiserum does HI test.
FJ11 plants of allantoic fluids sterile saline is made 1000 times of dilutions by 9.2, anti-with the anti-AIV H9 subtype sepcific of equivalent Serum mixing, sets in room temperature and after 1 hour, and allantoic cavity is inoculated with SPF chicken embryo 10, every embryo 0.2ml, while it is anti-to set H9 subtype sepcific Make 1000 with the control of NDV in serum with FJ11 plants of allantoic fluids and dilute each 10 not neutralized of control (inoculation 0.1ml).Observation And record result.
The preparation of embodiment 2:H9 subtype avian influenza antigen
1 inoculation takes production seed culture of viruses, makees appropriate dilution (such as 10 with sterile saline-3Or 10-4), it presses and " automatically connects Kind machine operation instruction " it requires, it is inoculated with the susceptible chicken embryo of 10~11 ages in days, every embryo 0.1ml seals pin hole after inoculation, sets 36~37 DEG C Continue to be incubated for, it is not necessary to turn over embryo.
After 2 are incubated for and observe egg inoculation, every sunshine embryo 1 time discards chicken embryo dead before 48 hours.Hereafter, every 4 ~6 hours photograph embryos 1 time, dead chicken embryo is taken out at any time, until 96 hours, no matter it is dead whether, all take out, gas chamber is straight upwards It is vertical, it is placed in 2~8 DEG C of coolings 12~24 hours.
3 harvests take out cooling chicken embryo, by " full-automatic cropper operation instruction " requirement, harvest chick embryo allantoic liquid (first It accepts orders for repairs or processing and receives dead germ after embryo).It draws blastochyle to be put in 50000ml sterilization container, sampling measurement erythrocyte agglutination valence.Agglutination titer is lower than 1:256 (micromethod) person should discard.Steriling test (can not transplant) is carried out by " Chinese veterinary pharmacopoeia " simultaneously, it should be without bacterial growth. It saves, should be no more than 5 at 2~8 DEG C before the blastochyle inactivation of harvest.
4 inactivations import H9 subtype avian influenza virus liquid in inactivation tank, and metered 10% formalin opens stirring Machine stirring, mixes them thoroughly, and the ultimate density of formaldehyde is 0.1%.It is imported in another inactivation tank after adding formalin, to avoid The virus that tank mouth nearby adheres to fails to contact inactivator.37 DEG C of inactivations (reach 37 DEG C of beginning timing with temperature in tank, open for 16 hours Blender is opened to continuously stir) it takes out afterwards, 2~8 DEG C of preservations are put, should be no more than 1 month.
5 inspection of semifinished product
5.1 steriling tests take the blastochyle of inactivation to test by existing " Chinese veterinary pharmacopoeia ", answer asepsis growth.
5.2 inactivations, which are examined, takes 10 age in days SPF chicken embryos 6, and inoculation inactivation concentration blastochyle, each 0.2ml are set in allantoic cavity 36~37 DEG C are continued to be incubated for, every sunshine embryo 2 times, are observed 5, are measured erythrocyte agglutination valence respectively to all blastochyles, should all not go out Now be aggregated, by a blastochyle harvest blind passage generation again, still without agglutination titer when, it is believed that inactivation is complete.
Blastochyle before inactivation is measured erythrocyte agglutination valence by the measurement of 5.3 erythrocyte agglutination valences, and agglutination titer is not less than 1:256 (micromethod) person, can be used for seedling.
The blastochyle taken out before inactivation is carried out 10 times and is serially diluted by the measurement of 5.4 viral levels, takes 10-6、10-7、10-83 Dilution, each allantoic cavity is interior to be inoculated with 10~11 age in days SPF chicken embryos 5, and every embryo 0.1m1 sets 36~37 DEG C and continues to be incubated for, every sunshine It embryo 2 times, observes 5, no matter dead germ, embryo living should all measure erythrocyte agglutination valence, and agglutination titer is not less than 1:16 (micromethod) person, sentences For infection, EID is calculated50, every viral level >=10 0.1ml7.3EID50When, seedling can be used for.
Embodiment 3: the preparation of vaccine
Mutually preparation takes 95 parts of mineral oil to 1 oil, 1 part of aluminum stearate, is uniformly mixed in oily phase preparation tank and is heated to 80 DEG C Afterwards, then 5 parts of Jia Siben -80, spare after cooling until temperature maintains 40 minutes when reaching 115 DEG C.
The preparation of 2 water phases mixes the H9 subtype avian influenza virus venom of inactivation with 1:2 ratio.95 parts of hybrid antigen liquid are taken, 5 parts of the Tween-80 of sterilizing, mixes well, is completely dissolved Tween-80.
3 emulsifications take 2 parts of oily phase to be put into emulsion tank, start motor, slow rotation stirring, while 1 part of water phase being added slowly Afterwards, then with 3500r/min stirring 30~40 minutes.After emulsification, takes vaccine 10ml to be added in centrifuge tube, be centrifuged with 3000r/min 15 minutes, the water phase that tube bottom is precipitated should be no more than 0.5ml.
4 packing quantitative separatings, seal, and adhesive label, set 2~8 DEG C of preservations.
5 product inspections
5.1 character
Appearance milky white emulsion.
Dosage form water-in-oil type.A cleaning suction pipe is taken, a small amount of vaccine is drawn and instills in cold water, in addition to the 1st drop, should all not be expanded It dissipates.
Stability is drawn vaccine 10ml and is added in centrifuge tube, with 3000r/min centrifugation 15 minutes, the water phase that tube bottom is precipitated 0.5ml should be not more than.
Viscosity is carried out by existing " Chinese veterinary pharmacopoeia " annex, should meet regulation.
5.2 loading quantity inspections are checked by existing " Chinese veterinary pharmacopoeia " annex, should meet regulation.
5.3 steriling tests are tested by existing " Chinese veterinary pharmacopoeia " annex, answer asepsis growth.
5.4 safety verifications vaccinate 1.0ml, observation 14 with 7~14 age in days SPF chickens 10, the subcutaneous branch of every neck Day, any locally and systemically adverse reaction as caused by vaccine should not occur.
5.5 efficacy test
5.5.1 serological method respectively subcutaneously or intramuscularly vaccinates 0.2ml with 30~60 age in days SPF chickens 15,10, Another 5 not immune to compare.21~28 days after inoculation, every chicken is taken a blood sample respectively, separate serum, by existing " Chinese veterinary pharmacopoeia " into The measurement of row HI antibody titer.The geometrical mean of immune group HI antibody titer should be not less than 1:180 (micromethod), non-immunized controls The geometrical mean of group HI antibody titer should be not higher than 1:4 (micromethod).
5.5.2 Immunization method respectively subcutaneously or intramuscularly vaccinates 0.2ml with 30~60 age in days SPF chickens 15,10, Another 5 not immune to compare.21~28 days after inoculation, every chicken wings 10 times of diluted YBF003 plants of venom of intravenous injection, every 0.2ml acquires cloacal swab 5 days after attacking poison, and after processing, allantoic cavity is inoculated with 10~11 age in days SPF chicken embryos 5, is incubated for Observation 5 days, no matter dead germ, embryo living should all measure chicken embryo liquid erythrocyte agglutination valence, in 5 chicken embryos of each swab samples inoculation only There is the agglutination titer of 1 chicken embryo liquid not less than 1:16 (micromethod), the virus purification positive can be judged to.Negative to virus purification Sample is determined again after answering blind passage primary.Immune group should 9 chicken virus purifications be at least feminine gender；Control group should at least 4 chickens Virus purification is the positive.
Embodiment 4: immunological cross protection test (can resist popular each place separation strains)
21 age in days SPF chickens, every 0.2ml is immunized in the inactivated vaccine prepared with FJ11 plants.21 days after immune, respectively with 10 Diluted FJ11 plants and the injection of each place isolated strain wing venous again, each strain respectively attacks 10 immune chickens and 5 same conditions Non- immunized controls chicken.After attacking poison, immune group chicken does not occur the typical clinical symptom of AI；Virus purification is carried out with cotton swab, Positive rate is no more than 10% (1/10)；And non-immunized controls group, also without typical clinical symptom, but virus purification is the positive. H9 subtype avian influenza inactivated vaccine prepared by showing FJ11 plants can resist the attack of FJ11 plants and each place separation strains and play Protective effect.Detailed results are shown in Table 1.
Moreover, comparative experiments shows to exempt from SPF chicken with the H9 subtype avian influenza inactivated vaccine sold currently on the market Epidemic disease, then challenge viral dosage is carried out with the FJ11 strain that the present invention screens；The result shows that the immune effect for the vaccine sold currently on the market Far below the immune effect of inactivated vaccine prepared by FJ11 strain of the invention；Be presumably due to FJ11 plants morph cause at present The immune effect of vaccine is bad.
Immunological cross protection test result of the table 1:FJ11 strain vaccine to different strains

Claims (3)

1. a kind of avian influenza virus, which is characterized in that the avian influenza virus is Avian Influenza Virus H9N2 FJ11 plants, Deposit number is CCTCC No.V201617.
2. avian influenza virus described in claim 1 is preparing the application in vaccine.
3. application as claimed in claim 2, which is characterized in that the vaccine is inactivated vaccine.
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CN104560890A (en) * 2014-12-03 2015-04-29 北京市兽医生物药品厂 H9 subtype avian influenza virus and application thereof
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CN104940921B (en) * 2015-07-07 2018-08-14 青岛易邦生物工程有限公司 A kind of H9 subtype avian influenza virus inactivated vaccines comprising chicken alpha-interferon albumen
CN104946600B (en) * 2015-07-07 2018-08-24 青岛易邦生物工程有限公司 A kind of H9 subtype avian influenza virus strain

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