CN104928259A - H9 subtype of avian influenza virus inactivating vaccine and preparation method thereof - Google Patents
H9 subtype of avian influenza virus inactivating vaccine and preparation method thereof Download PDFInfo
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Abstract
The invention discloses an H9 subtype of avian influenza virus inactivating vaccine and a preparation method thereof. The H9 subtype of avian influenza virus inactivating vaccine contains an inactivated H9 subtype of avian influenza virus SY strain. The preparation method of the H9 subtype of avian influenza virus inactivating vaccine comprises the following steps of (1) preparing an H9 subtype of avian influenza virus SY strain liquid; (2) inactivating the H9 subtype of avian influenza virus SY strain liquid; (3) preparing an oil-phase solution; (4) preparing an aqueous phase solution; (5) emulsifying the vaccine to prepare the avian influenza inactivating vaccine. The inactivating vaccine is good in immunogenicity, is high in antibody producing level, and is long in lasting period; the adopted preparation method is simple and safe.
Description
Technical field
The invention belongs to technical field of animal virology, be specifically related to a kind of H9 subtype avian influenza virus, inactivated vaccine and preparation method thereof.
Background technology
Avian influenza virus (Avian Influenza virus, AIV) is under the jurisdiction of orthomyxovirus section influenza A and belongs to, and in polymorphism, wherein spherical diameter 80 ~ 120nm, has cyst membrane.Genome is segmented sub-thread strand RNA.Multiple bird can be infected, also can infect the mankind.According to its adventitia hemagglutinin (Haemagglutinin, and neuraminidase (Neuraminidase HA), NA) difference of protein antigenicity is divided into different hypotypes, comprise 16 kinds of HA hypotypes and 10 kinds of NA hypotypes, both various combinations form the avian influenza virus of different subtype.The antigenic drift that both HA and NA had due to AIV occur or transfer, AIV is easy to morph, and constantly breaks through the kind obstacle of its infection host, causes new influenza pandemic.Since being separated to H9N2 hypotype AIV from 1966 first in the turkey body of North America, this subtype virus is worldwide propagated rapidly, within 1988, China Hong Kong is separated to Asia first strain H9N2 hypotype AIV, China mainland infects in reported first chicken group in 1992, within 1994, is separated to this hypotype AIV first.
H9N2 subtype avian influenza belongs to low pathogenicity bird flu, and sickness rate is high, but mortality ratio is low.Main clinical manifestation is only slight respiratory symptom, appetite reduces, laying rate can be down to less than 20% from 90%, even have no harvest, the death of about 30% can be caused to commercial meat bird, very easily with intestinal bacteria polyinfection, have a strong impact on the production performance of poultry, provisions fowl industrial belt carrys out serious financial loss.The more important thing is, this hypotype AIV can pass through host's obstacle infection Mammals and comprise people, also has an opportunity to be very popular the mankind, brings serious threat to human health.Current H9N2 hypotype is the AIV Main Subtype existed in China chicken group, accounts for more than 90% of bird flu total incidence.In various prevention and control measure, vaccine immunity is still most important measure.At present, although the widespread use of H9 subtype avian influenza inactivated vaccine, this disease have also been obtained effective control, and H9 subtype avian influenza still happened occasionally in vaccinated flock in recent years.For this reason, filter out a strain immunogenicity by research strong, the vaccine strain of immunoprotection can be provided particularly necessary for homology/non-homogeneous H9 hypotype AIV epidemic strain.
Summary of the invention
Technical problem to be solved by this invention is for above-mentioned the deficiencies in the prior art, provides a kind of H9 subtype avian influenza virus, inactivated vaccine and preparation method thereof.This inactivated vaccine immunogenicity is good, and produce antibody horizontal high, the extended period is long; The preparation method adopted is simple, safety.
For solving the problems of the technologies described above, the technical solution used in the present invention is, a kind of H9 subtype avian influenza virus SY strain, its deposit number is CGMCC No.5968.
Present invention also offers the application of a kind of H9 subtype avian influenza virus SY strain in preparation prevention H9 subtype avian influenza vaccine.
Present invention also offers the application of a kind of H9 subtype avian influenza virus SY strain in preparation H9 subtype avian influenza diagnostic reagent and medicine.
Present invention also offers a kind of H9 subtype avian influenza inactivated vaccine, it contains the H9 subtype avian influenza virus SY strain through deactivation.
Present invention also offers a kind of preparation method of H9 subtype avian influenza inactivated vaccine, comprise as follows:
(1) prepare H9 subtype avian influenza virus SY strain venom: produce seed culture of viruses with AIV SY strain and inoculate healthy susceptible chicken embryo, results infected chicken blastochyle is as H9 subtype avian influenza virus SY strain venom;
(2) deactivation H9 subtype avian influenza virus SY strain venom: add formaldehyde solution in the H9 subtype avian influenza virus SY strain venom of the middle gained of step (1);
(3) preparation of oil-phase solution: by injection white oil and Si Ben-80 according to volume ratio 94:6 mixing, then add aluminum stearate 2 parts, with heating be stirred to transparent till, autoclaving obtains oil-phase solution;
(4) preparation of aqueous phase solution: the H9 subtype avian influenza virus SY strain venom of deactivation in step (2) and the tween-80 of sterilizing are mixed according to volume ratio 96:4, obtains aqueous phase solution;
(5) vaccine emulsification: the aqueous phase solution in the oil-phase solution in step (3) and step (4) is even according to volume ratio 1.5 ~ 2.3:1 ratio mixing and emulsifying, makes inactivated avian influenza vaccine.
Further, the H9 subtype avian influenza virus SY strain venom in this step (1), refers to the embryo chicken blastochyle alive of rear 24 ~ 96 hours dead chicken embryos of inoculation and 96 hours, viral level>=10 in every 0.1mlH9 subtype avian influenza virus SY strain venom
8.0eID
50.
Further, in SY strain venom, the mass concentration of formaldehyde is 0.1 ~ 0.2% in this step (2), and deactivation 24 hours under the condition of 37 DEG C.
A kind of H9 of the present invention subtype avian influenza virus, inactivated vaccine and preparation method thereof have following advantage:
1. this H9 subtype avian influenza virus SY strain, has stable, good specificity and immunogenicity, can be used for preparation H9 subtype avian influenza vaccine, diagnostic reagent and medicine.
2., after the H9 subtype avian influenza inactivated vaccine inoculation prepared by, antibody generation time is short, height of tiring, and immune duration is long, all has stronger immunoprophylactic effect to homology/nonhomologous H9 subtype avian influenza virus epidemic strain.
3. the adopted method preparing H9 subtype avian influenza inactivated vaccine is simple, safety, cost are low.
H9 subtype avian influenza virus SY strain in the present invention, that the dead chicken pathological material of disease of clinical doubtful avian influenza is separated to a strain Lowly Pathogenic Avian Influenza Virus from somewhere, Shaanxi Province, identify through veterinary institute state poultry influenza research centre, Harbin, for H9N2 subtype avian influenza virus SY strain, called after A/Chicken/Shaanxi/SY/97 (H9N2) strain, Classification And Nomenclature: influenza A H9N2 hypotype.
Preservation information is as follows:
Depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center;
Unit address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica;
Preservation date: on April 11st, 2012;
Deposit number: CGMCC No.5968.
In the present invention, H9 subtype avian influenza virus SY strain has following characteristics:
1. coagulation: by " Chinese veterinary pharmacopoeia " (the Chinese veterinary pharmacopoeia council. People's Republic of China (PRC) veterinary drug allusion quotation two 〇 mono-〇 version three, Chinese agriculture press, 2011) method carry out.1:1024 (micromethod, lower same) is not less than to chicken red blood cell agglutination titer.The erythrocytic characteristic of this viral agglutination can be suppressed by bird flu H9 positive serum specificity, can not be suppressed by newcastle disease (ND), egg drop syndrome (EDS) and bird flu H5 positive serum.
2. specificity: by viral dilution to containing 10
5.0eID
50/ 0.1ml, mixes with the anti-H9 subtype avian influenza virus specific serum of equivalent, and in room temperature and after 1 hour, inoculation SPF chicken embryo 10, observe 96 hours, do not cause specificity dead and at least survive 8 in 24 ~ 96 hours, the hemagglutination test (HA test) of chicken blastochyle is negative.
3. intravenous inoculation pathogenic index (IVPI) measures: measure by " People's Republic of China's veterinary biologics quality standard " two 〇 〇 1 year version annex method, IVPI≤0.04.
4. pure property: carry out bacterium, mould, mycoplasma and exogenous virus inspection by " Chinese veterinary pharmacopoeia " annex method, the results are shown in Table 1, be feminine gender.
5. viral passages: virus sterile saline is done 10000 times of dilutions, inoculation 10 age in days SPF chicken embryos in allantoic cavity, every embryo 0.1ml, puts 37 DEG C and hatches.Collect inoculation death in latter 72 ~ 96 hours and the obvious chicken embryo of pathology, results infected chicken blastochyle, is loaded in sterile chamber.By inspection aseptic and to 1% chicken red blood cell agglutination titer be not less than 1:512 chicken blastochyle mixing, quantitative separating, indicate harvest date, Virus passages etc., according to said method by AIV SY strain primary viral in the continuous passage of SPF chicken embryo to the 15th generation.
6. viral level measures: each generation virus sterile saline is made 10 times of serial dilutions, gets 10
-7, 10
-8, 10
-93 extent of dilution, inoculation 10 age in days SPF chicken embryo 5 pieces in each allantoic cavity, every embryo 0.1ml, put 37 DEG C to continue to hatch, chicken embryo dead before 24 hours discards to be disregarded, and the chicken embryo dead at 24 ~ 96 hours takes out at any time, results chicken blastochyle, same dilution blastochyle balanced mix, measures red cell agglutination valency respectively by extent of dilution, to 96 hours, take out all embryos alive, gather in the crops chicken blastochyle one by one, measure red cell agglutination valency respectively, agglutination titer is not less than 1:32 person and is judged to infection, calculates EID
50.Every 0.1ml viral level is 10
8 . 25~ 10
8 . 65eID
50between.The results are shown in Table 1:
Table 1 AIV SY strain each generation viral level, pure property and HA titration result
Note: "-" represents feminine gender.
7. pathogenic:
(1) to the virulence of chicken embryo: by AIV SY strain the 3rd generation, 5 generations, 10 generations, 15 generation virus do 10000 times of dilutions with sterile saline respectively, inoculation 10 age in days SPF chicken embryo 10 in allantoic cavity, every embryo 0.1ml, put 37 DEG C hatch 96 hours after observe pathology, the pathologies such as result has 8 chicken embryo deaths at least, and idiosome performance severe is congested, hemorrhage.
(2) to the virulence of chick: by AIV SY strain virus vena axillaris injection SPF chicken in 7 week age 10, in attacking poison latter 3rd ~ 15 days, gather larynx and the cloacal swabs of every chicken every day, both are mixed rear filtration sterilization, and inoculate 5 piece of 10 age in days SPF chicken embryo through allantoic cavity, every piece of chicken embryo 0.2ml, be placed in 37 DEG C of incubators and hatch, discard the dead germ before 24 hours, gather in the crops blastochyle that is dead at 24 ~ 96 hours and 96 hours survival chicken embryos one by one, measure red cell agglutination valency respectively, as long as have the blastochyle red cell agglutination valency of 1 piece of chicken embryo to be not less than 1:16 in the chicken embryo of every part of cotton swab sample inoculation, namely corresponding test chicken virus purification is sentenced for positive.Primary virus is separated negative sample, judges after a blind passage generation again.Result virus purification situation is as table 2.Namely SPF chicken starts toxin expelling on the 3rd day after infecting, toxin expelling peak period is after attacking poison 4 ~ 7 days (test chicken 10/10 outwardly toxin expelling), to attacking poison latter 14 days test chickens no longer toxin expelling, and test chicken attacks poison, and outwardly the shortest time of toxin expelling is 5 days continuously afterwards, and maximum duration is 11 days.
Table 2 AIV SY strain is to SPF chicken pathogenic measurement result
Note: "+" represents virus purification for positive, and "-" represents feminine gender.
8. immunogenicity: by AIV SY strain the 5th generation, 10 generations, 15 generation virus liquid, make deactivation vaccine respectively by the inventive method, if 0.1ml, 0.3ml, 0.5ml/ dosage group, respectively immunity SPF chicken in 4 weeks age, 10/group, 10 contrast chickens and do not inoculate.Inoculate latter 7 days, 14 days, 21 days to take a blood sample respectively, separation of serum, measure AI HI antibody; Simultaneously in exempting from latter 21 days intravenous injection AIV SY strain (10
8.7eID
50), attack malicious latter 5th day from test chicken larynx and cloacal swab isolated viral, statistics virus isolated rate.The results are shown in Table 3.Within 7 days, can detect HI antibody after the vaccination of immunity chicken, within 14 days, respectively organize immune chicken HI antibody geometrical mean (GMT) all at more than 1:64, respectively organize immune chicken antibody GMT and all reach more than 1:256 when 21 days, contrast chicken antibody level is negative entirely.And immune chicken is not all separated to virus after attacking poison, contrast chicken virus isolated rate 9/10, result absolutely proves that avian influenza virus SY strain has good immunogenicity within 15 generations, can use as vaccine strain.
Table 3 different generation AIV SY strain immunogenicity detected result
Note: "-" represents feminine gender.
Embodiment
SPF chicken embryo used in the embodiment of the present invention is purchased from Beijing Cimmeria Wei Tong laboratory animal Technology Co., Ltd..Bird flu H5, H7, H9 positive serum is purchased from Harbin Weike Biologic Technology Ltd., and newcastle disease (ND), egg drop syndrome (EDS) positive serum are purchased from China Veterinery Drug Inspection Office.
Embodiment 1
The separation andpreconcentration of virus:
1. virus purification: by the tissue such as spleen, liver, heart of dead for the doubtful bird flu of clinical censorship chicken, sterile saline grinding is added in 1:5 (W/V) ratio after weighing, then freeze thawing 3 times, 3000 revs/min centrifugal 30 minutes, 10 age in days SPF chicken embryo 10 pieces is inoculated after supernatant liquor sterile filtration, wherein 7 pieces of allantoic cavity inoculation pathological material of disease process supernatant liquor 0.1ml/ piece, another 3 pieces of inoculation physiological saline compare.After inoculation, chicken embryo is put back in incubator and continue to hatch, every day shines egg 2 times, discard dead chicken embryo in 24h, chicken embryo dead after 24h is taken out in time, puts 4 DEG C of refrigerator 4h, more asepticly collect chick embryo allantoic liquid, and observe the characteristics of lesion of chicken embryo, not dead embryo is observed to 96h, takes out and collects chick embryo allantoic liquid by upper method, and sampling detects.
2. hemagglutination test (HA) and hemagglutination-inhibition test (HI): undertaken by the method for " Chinese veterinary pharmacopoeia ".Results chicken blastochyle can aggegation chicken red blood cell, and HA valency is 1:1024.And the hemagglutination activity of this unknown virus can be suppressed by bird flu H9 positive serum specificity, can not be suppressed by ND, EDS and bird flu H5 positive serum.
3. viruses indentification: unknown virus allantoic fluid is carried out phospho-wolframic acid negative staining, electric Microscopic observation after suitably diluting, there is under mirror the virus particle of typical orthomyxovirus morphological specificity as seen.Allantoic fluid sample is sent veterinary institute state poultry influenza research centre, Harbin simultaneously, after serological method qualification, this isolate is H9N2 subtype avian influenza virus, called after A/Chicken/Shaanxi/SY/97 (H9N2) strain (being called for short SY strain).
4. intravenous inoculation pathogenic index (IVPI) measures: specify method by International Office of Epizootics (OIE), 10 times will be diluted containing viral allantoic fluid sterile saline, by this virus dilution liquid with 0.1ml/ plumage intravenous inoculation 10 SPF chicken in 6 week age, chicken group is checked once every 24 hours, observe 10 days altogether, each observation is given a mark to chicken, normal chicken meter does 0, sick chicken meter does 1, grave illness chicken meter does 2, 3 (the following a kind of symptoms of sick chicken performance made by dead chicken meter, grave illness chicken then shows multiple symptom, as respiratory symptom, depressed, diarrhoea, without fur skin and wattle cyanosis, face or head swelling, nervous symptoms).Result is as table 4:
Table 4 IVPI measurement result
IVPI＝4/100＝0.04。
Morbidity chicken show as right eye swelling, rubescent, can't keep one's eyes open, to transference cure when the 5th day, recover normal.
5. viral level measures: viral allantoic fluid sterile saline is made 10 times of serial dilutions, gets 10
-7, 10
-8, 10
-9, 10
-104 extent of dilution, allantoic cavity inoculates each 7 of 10 age in days SPF chicken embryo respectively, every embryo 0.1ml, observe to 96 hours, chicken embryo dead in 24 hours discards to be disregarded, within 24 ~ 96 hours, dead chicken embryo takes out at any time, results chicken blastochyle, same dilution blastochyle balanced mix, measures red cell agglutination valency respectively by extent of dilution, embryo of living after 96 hours, gather in the crops blastochyle one by one, measure red cell agglutination valency respectively, chicken embryo death, idiosome, head, pawl, wing are hemorrhage, liver is rubescent or allantoic fluid HA tires is judged to infection at more than 1:32, calculates EID
50, EID
50=10
-8.24/ 0.1ml.
Embodiment 2
H9 subtype avian influenza inactivated vaccine immuning effect test:
This H9 subtype avian influenza inactivated vaccine contains the H9 subtype avian influenza virus SY strain through deactivation.Its preparation method is as follows:
(1) H9 subtype avian influenza virus SY strain venom is prepared: produce seed culture of viruses with AIV SY strain and inoculate healthy susceptible chicken embryo, results infected chicken blastochyle is as H9 subtype avian influenza virus SY strain venom, wherein, H9 subtype avian influenza virus SY strain venom, refer to the embryo chicken blastochyle alive of rear 24 ~ 96 hours dead chicken embryos of inoculation and 96 hours, viral level>=108.0EID in every 0.1mlH9 subtype avian influenza virus SY strain venom
50;
(2) deactivation H9 subtype avian influenza virus SY strain venom: add formaldehyde solution in the H9 subtype avian influenza virus SY strain venom of the middle gained of step (1), to make the mass concentration of formaldehyde in SY strain venom for 0.1 ~ 0.2%, and deactivation 24 hours under the condition of 37 DEG C;
(3) preparation of oil-phase solution: by injection white oil and Si Ben-80 according to volume ratio 94:6 mixing, then add aluminum stearate 2 parts, with heating be stirred to transparent till, autoclaving obtains oil-phase solution;
(4) preparation of aqueous phase solution: the H9 subtype avian influenza virus SY strain venom of deactivation in step (2) and the tween-80 of sterilizing are mixed according to volume ratio 96:4, obtains aqueous phase solution;
(5) vaccine emulsification: the aqueous phase solution in the oil-phase solution in step (3) and step (4) is even according to volume ratio 1.5 ~ 2.3:1 ratio mixing and emulsifying, makes inactivated avian influenza vaccine.
1.H9 subtype avian influenza inactivated vaccine serology efficacy test and Immunization protect the test of dependency:
Get 40 21 age in days SPF chickens, respectively with 0.05ml, 0.07ml, 0.1ml tri-dosage neck subcutaneous vaccination inactivated avian influenza vaccines, each dose inoculation 10, does not inoculate in contrast for another 10.Blood sampling in 3 weeks after immunity, every equal marker number of chicken, detects AI HI antibody horizontal in each chicken serum, then according to the height of antibody horizontal, again divided into groups by test chicken, the chicken that antibody horizontal is identical or close is divided into one group, and every chicken intravenous injection AIV SY strain 0.2ml is (containing 10
8.0eID
50) carry out challenge test, within 5th, gather larynx and the cloacal swab of every chicken after attacking poison respectively, isolated viral, adds up the virus isolated rate (attacking malicious protection ratio) of each antibody horizontal group.
Test-results is in table 5: as can be seen from Table 5; in test chicken serum AI HI antibody horizontal height and there is certain parallel relation between protection ratio after attacking poison; as HI antibody titer≤1:2; attacking malicious protection ratio is 0, when reaching 1:16, and attacking malicious protection ratio is 33.3%; when reaching 1:32; attacking malicious protection ratio is 80%, and as antibody horizontal >=1:64, attacking the rear protection ratio of poison is 100%.Therefore, when carrying out the efficacy test of H9 subtype avian influenza inactivated vaccine, serological method and Immunization Fa Keren select one.
Table 5 chicken antibody level and protest test result
2.H9 subtype avian influenza inactivated vaccine duration of immunity is studied; Its test method is as follows:
(1) 3 week SPF chicken immune phase in age tested: by 3 batches of H9 subtype avian influenza inactivated vaccines respectively with 0.3ml/ dosage only, neck subcutaneous vaccination SPF chicken in 3 week age, every dosage group 10.Latter 7th, 14,21,30,60,90,120,150 day of immunity, together with contrast chicken 5, takes a blood sample respectively, detects serum AI HI antibody.
(2) 7 week SPF chicken immune phase in age tested: by 3 batches of H9 subtype avian influenza inactivated vaccines respectively with 0.5ml/ dosage only, and grouping immunity SPF chicken in 7 week age, often organizes 10.Before exempting from and after immunity the 1st, 2,3,4,5,6,7 months, together with contrast chicken 5, take a blood sample respectively, detect serum AI HI antibody.
(3) immune period test of antenatal laying hen is opened: by 3 batches of H9 subtype avian influenza inactivated vaccines respectively with 0.5ml/ immunizing dose only, hen in antenatal 18 week age (northwest agricultural section macrospecies chicken house Roman egghen) is opened in grouping immunity, often organizes 10.Before immunity with immunity after the 1st, 2,3,4, May, together with contrast chicken 5, take a blood sample respectively, detect serum AI HI antibody.
Test-results is in table 6, table 7, table 8, and to 3 week age, the duration of immunity of SPF chicken at least can reach 5 months.To 7 week age, the duration of immunity of SPF chicken at least can reach 7 months.3 batches of Combined vaccine split the immune period test of antenatal laying hen, and the duration of immunity of 3 batches of Combined vaccine at least can reach 5 months.
Table 6 is to the immune period test of SPF chicken in 3 week age
Table 7 is to the immune period test of SPF chicken in 7 week age
Table 8 is to the immune period test of Roman egghen in 18 week age
Protest test to H9 hypotype AIV epidemic isolates after the immunity of 3.H9 subtype avian influenza inactivated vaccine:
To 21 age in days SPF chicken neck dorsal subcutaneous injection H9 subtype avian influenza inactivated vaccine 0.3ml/ only, inoculate blood sampling in latter 21 days, detect serum AIV HI antibody titer, and use AIV SY strain and AIV epidemic isolates Hz, Gz, Qd, Fj etc. (the local epidemic isolates of H9 hypotype AIV that the ground such as Shaanxi, Qingdao, Fujian is separated) to attack poison respectively, within 5th, swab sample is adopted from larynx and cloaca, with 10 age in days SPF chicken embryo isolated virals after attacking poison.Result is as table 9, and immune group chicken antibody level is all at more than 1:400, and virus purification is feminine gender.Therefore, the attack of the H9 subtype avian influenza virus be separated from different areas can be resisted with the chicken of inactivated avian influenza vaccine (SY strain) immunity, can effectively prevent H9 subtype avian influenza.
The challenge test of table 9 bird flu H9 hypotype epidemic isolates
Note: the positive chicken number of * virus purification/attack malicious chicken number.
Claims (7)
1. a H9 subtype avian influenza virus SY strain, its deposit number is CGMCC No.5968.
2. the application of a H9 subtype avian influenza virus SY as claimed in claim 1 strain in preparation prevention H9 subtype avian influenza vaccine.
3. the application of a H9 subtype avian influenza virus SY as claimed in claim 1 strain in preparation H9 subtype avian influenza diagnostic reagent and medicine.
4. a H9 subtype avian influenza inactivated vaccine, is characterized in that containing the H9 subtype avian influenza virus SY strain through deactivation.
5. a preparation method for H9 subtype avian influenza inactivated vaccine as claimed in claim 4, is characterized in that, comprise as follows:
(1) prepare H9 subtype avian influenza virus SY strain venom: produce seed culture of viruses with AIV SY strain and inoculate healthy susceptible chicken embryo, results infected chicken blastochyle is as H9 subtype avian influenza virus SY strain venom;
(2) deactivation H9 subtype avian influenza virus SY strain venom: add formaldehyde solution in the H9 subtype avian influenza virus SY strain venom of the middle gained of step (1);
(3) preparation of oil-phase solution: by injection white oil and Si Ben-80 according to volume ratio 94:6 mixing, then add aluminum stearate 2 parts, with heating be stirred to transparent till, autoclaving obtains oil-phase solution;
(4) preparation of aqueous phase solution: the H9 subtype avian influenza virus SY strain venom of deactivation in step (2) and the tween-80 of sterilizing are mixed according to volume ratio 96:4, obtains aqueous phase solution;
(5) vaccine emulsification: the aqueous phase solution in the oil-phase solution in step (3) and step (4) is even according to volume ratio 1.5 ~ 2.3:1 ratio mixing and emulsifying, makes inactivated avian influenza vaccine.
6. the preparation method of a H9 subtype avian influenza inactivated vaccine as claimed in claim 5, it is characterized in that, H9 subtype avian influenza virus SY strain venom in described step (1), refer to the embryo chicken blastochyle alive of rear 24 ~ 96 hours dead chicken embryos of inoculation and 96 hours, viral level>=10 in every 0.1mlH9 subtype avian influenza virus SY strain venom
8.0eID
50.
7. a preparation method for H9 subtype avian influenza inactivated vaccine as claimed in claim 5, is characterized in that, in SY strain venom, the mass concentration of formaldehyde is 0.1% ~ 0.2% in described step (2), and deactivation 24 hours under the condition of 37 DEG C.
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| CN113151189A (en) * | 2020-12-29 | 2021-07-23 | 江苏省农业科学院 | H9N2 avian influenza virus, inactivated vaccine and preparation method thereof |
| CN116875564A (en) * | 2023-09-07 | 2023-10-13 | 天津瑞普生物技术股份有限公司 | H9 subtype avian influenza virus strain and culture method thereof |
| CN116875564B (en) * | 2023-09-07 | 2023-12-01 | 天津瑞普生物技术股份有限公司 | H9 subtype avian influenza virus strain and culture method thereof |
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