CN104560890A - H9 subtype avian influenza virus and application thereof - Google Patents
H9 subtype avian influenza virus and application thereof Download PDFInfo
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- CN104560890A CN104560890A CN201410727960.8A CN201410727960A CN104560890A CN 104560890 A CN104560890 A CN 104560890A CN 201410727960 A CN201410727960 A CN 201410727960A CN 104560890 A CN104560890 A CN 104560890A
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Abstract
The invention discloses an H9 subtype avian influenza virus, an H9 subtype avian influenza inactivated vaccine and a preparation method thereof. The H9 subtype avian influenza virus has higher immunogenicity and better cross protection performance. The H9 subtype avian influenza inactivated vaccine prepared from the H9 subtype avian influenza virus has better immunoprotection effects.
Description
Technical field
The present invention relates to biological pharmacy technical field, relate to H9 subtype avian influenza virus and H9 subtype avian influenza inactivated vaccine and preparation method thereof.
Background technology
Bird flu (Avian influenza, AI) is the infectious disease of a kind of serious harm avian health caused by influenza A.After birds infects bird flu virus (Avian influenza virus, AIV), symptom can show as inapparent infection, Asia is faced and examined infection, or slight respiratory tract disease, egg production reduce until various ways such as acute systemic fatal diseases.According to the difference of AIV to susceptible chicken pathogenic, AIV can be divided into highly pathogenic AIV and low pathogenicity AIV.
Hommee in 1966 etc. suffer from the turkey of gentle respiratory tract disease from the Wisconsin of U.S. the north and have been separated to a strain AIV-A/Turkey/Wisconsin/1/66, and be accredited as H9 hypotype through serology, this is first separated strain of H9 hypotype AIV.Afterwards, in the world, other places are found H9N2AIV in succession, and in pandemic situation, cause serious economic loss to aviculture.The Bennejean of France was at 1979 and 1980, the bird flu virus H9N2 that can cause inferior clinical symptom is separated in chicken body, within 1978 and 1979, in the turkey of U.S.'s morbidity, be separated to H9N2AIV, the reports such as Alexander, Nagarajan in 2009 etc. have also been separated to H9N2AIV from India's chicken house.The AI explosion facies secondary that 1994-1999 is caused by H9N2 AIV is born in all over the world, comprises the Ireland in Europe, Germany, Italy, Iran in Asia, Pakistan, Saudi Arabia, Israel, Korea S, China and the ground such as the U.S. and South Africa.
H9N2 AIV is in the popularity of China.Shortridge (1992) was as far back as 1975 to 1985, just long-term Influenza Surveillance is carried out to the birds in live-bird market, Hong Kong, in the duck body of appearance health, be separated to H9 hypotype AIV, but but in chicken body, do not find the AIV of this hypotype.Within 1994, Guangdong Province's chicken house Egg Production of Laying Hens declines 14% ~ 75%, broiler mortality rate is 10% ~ 40%, Chen Bailun etc. have been separated to 6 strain virus in clinical disease chicken body, serological Identification is H9 hypotype, this is that in China, ground is separated to H9 hypotype AIV first, by these Viral experiment rooms inoculation chicken, shows as typical clinical symptoms, but rehabilitation soon, and unlikely chicken death.1996, Tang Xiuying etc. were separated to 1 strain H9N2 virus from Sichuan morbidity chicken group, and Chen Fuyong etc. are also separated to H9N2 AIV from North China, and clone the gene order of its nucleoprotein and analyze.In recent years again from the morbidity chicken group of China some areas, duck group, Carnis Coturnicis japonicae body, be separated to many strains H9N2 AIV, by carrying out finding in bird flu serosurvey to part province of China, city, district's commodity egg chicken house and poultry village specializing in a certain trade, the positive chicken group of H9 hypotype accounts for total AIV antibody positive chicken group's 93.67%, wherein the overwhelming majority is H9 hypotype, illustrate that H9 hypotype AIV extensively exists in China, be the Main Subtype of the existing AIV of China, huge economic loss is caused to the development of China's aviculture.
Because bird flu virus makes a variation quickly, bring difficulty to safety control of bird flu.Current vaccination is one of Main Means of prevention and control of fowl influenza, and because bird flu virus variation is fast, epidemic isolates is many, and serology is many and without cross immunity, its effect is restricted greatly.
As can be seen here, need immunogenicity stronger in this area, intersecting protective better vaccine H9 subtype avian influenza virus strain.
Summary of the invention
The object of the present invention is to provide a kind of H9 subtype avian influenza virus with stronger immunogenicity and better intersecting protective, H9 subtype avian influenza inactivated vaccine prepared therefrom and preparation method thereof.
For reaching this object, the present invention by the following technical solutions:
In first aspect, the invention provides a kind of H9 subtype avian influenza virus, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), preservation date is on October 22nd, 2010, and deposit number is CGMCC No.9813.
In second aspect, the invention provides a kind of H9 subtype avian influenza inactivated vaccine, described vaccine comprises the H9 subtype avian influenza virus as described in relation to the first aspect of deactivation.
In the third aspect, the invention provides a kind of preparation method of H9 subtype avian influenza inactivated vaccine, comprise the following steps:
A () culture fluid to the H9 subtype avian influenza virus comprised described in first aspect carries out deactivation, to obtain the solution containing deactivation H9 subtype avian influenza virus;
B () adds emulsifying agent to the described solution containing deactivation H9 subtype avian influenza virus and prepares aqueous phase; And
C described aqueous phase and oil phase are carried out mixing and emulsifying by (), to obtain described H9 subtype avian influenza inactivated vaccine
In the preparation method of H9 subtype avian influenza inactivated vaccine of the present invention, before step (a), described method can also comprise to be inoculated in nonimmune Embryo Gallus domesticus by H9 subtype avian influenza virus as described in relation to the first aspect, and at 35.5 ~ 37.5 DEG C, preferred 36.0 ~ 37.0 DEG C, cultivate at preferred 36.5 DEG C, the chick embryo allantoic liquid that collection obtains also collects supernatant to obtain the culture fluid comprising H9 subtype avian influenza virus as described in relation to the first aspect by continuous flow centrifugation, and avian influenza venom hemagglutination test HA imitates valency≤2
8, and malicious valency ELD (1:256)
50≤ 10
8, can use.
In the preparation method of H9 subtype avian influenza inactivated vaccine of the present invention, deactivation described in step (a) can be included in 2 DEG C ~ 8 DEG C, under preferred 4 DEG C of-6 DEG C of conditions, utilize final concentration for 1:2000 ~ 1:4000, preferred 1:2500 ~ 1:3500, more preferably the culture fluid of propiolactone to the described H9 of comprising subtype avian influenza virus of 1:3000 carries out inactivation treatment 16 ~ 36 hours, preferred 20-30 hour, preferred 25 hours; And propiolactone was hydrolyzed in 2 hours 37 DEG C of Water Under solutions, to obtain the solution containing deactivation H9 subtype avian influenza virus.
In the preparation method of H9 subtype avian influenza inactivated vaccine of the present invention, preparing aqueous phase and can comprise the described solution containing deactivation H9 subtype avian influenza virus is mixed with the volume ratio of tween 80 according to 94 ~ 97:3 ~ 6, to obtain aqueous phase described in step (b); Wherein, the concentration of described tween 80 is 3.0%.
In the preparation method of H9 subtype avian influenza inactivated vaccine of the present invention, the preparation of described oil phase can comprise medical grade white oil (MARCAL-52, Mobil), Si Ben-80 and aluminium stearate mix according to the mass ratio of 94:6:2, to obtain described oil phase.
In the preparation method of H9 subtype avian influenza inactivated vaccine of the present invention, in step (c), described aqueous phase and oil phase being carried out that mixing and emulsifying can to comprise described aqueous phase and oil phase according to volume ratio is that 2:3 ~ 2:4 carries out mixing and emulsifying, to obtain described H9 hypotype disease vaccine.
In fourth aspect, the invention provides the application of H9 subtype avian influenza virus in the preparation of the medicine of prevention and therapy H9 subtype avian influenza as described in relation to the first aspect.
In the 5th, the invention provides the application of H9 subtype avian influenza inactivated vaccine in the preparation of the medicine of prevention and therapy H9 subtype avian influenza as described in second aspect.
Advantageous Effects of the present invention is: (specifically seeing embodiment 4)
1, compared with existing H9 subtype avian influenza virus, H9 subtype avian influenza virus immunogenicity of the present invention is stronger and intersecting protective is better.Specifically see embodiment 4.
2, compared with existing H9 subtype avian influenza virus vaccine, H9 subtype avian influenza virus vaccine of the present invention has better immune protective effect.Specifically see embodiment 4.
Preservation explanation
Classification And Nomenclature: Avian Influenza Virus H9N2
Preservation mechanism: China Committee for Culture Collection of Microorganisms's common micro-organisms center
Preservation organization address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City
Preservation date: on October 22nd, 2014
Register on the books numbering in preservation center: CGMCC No.9813
Detailed description of the invention
Technical scheme of the present invention is further illustrated below by detailed description of the invention.
The separation andpreconcentration of embodiment 1H9 subtype avian influenza virus (HD strain)
1, virus purification
2012 there is infectious disease in Jiangsu broiler breeding factory, collection live-bird or frequently the time of death sample comprise trachea, cloacal swab, liver, spleen etc., by collected specimens homogenate in high-speed homogenization machine, in the isotonic phosphate buffer liquid (PBS) added according to the ratio of 1:5.Sample liquid is through 1, the centrifugal 10min of 500r/min, get supernatant 0.22 μm of germ tight filter to filter, the sterile tissue sample handled well is inoculated 9 ~ 11 age in days SPF Embryo Gallus domesticus 0.2mL/ piece through chorioallantoic cavity, hatch in 36.5 ~ 38.5 DEG C, every 6h, according to an embryo, discards nonspecific death embryo in 24h, collect the dead embryo of 24 ~ 96h and the not dead embryo allantoic liquid of 96h, place 2 DEG C ~ 8 DEG C cold embryos for subsequent use.
2, hypotype qualification
Serum neutralization test (HI experiment) is utilized to carry out hypotype qualification: carry out on V-type hemagglutination test micro plate by micromethod, chicken red blood cell concentration is 1% (v/v), and reaction total amount is 0.075mL.Identified according to the method described above by the HD strain chick embryo allantoic liquid of separation, qualification result proves that the strain be separated is H9 subtype avian influenza virus, and called after A/Chicken/Jiangsu/12/2013 (H9N2 hypotype) strain is called for short HD strain.This strain is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center (being called for short CGMCC) preservation on October 22nd, 2014, and Classification And Nomenclature is bird flu virus H9 hypotype, and preserving number is CGMCC No.9813.Qualification standard serum and standard antigen army are purchased from state of Harbin Veterinary Medicine Inst., China Academy of Agriculture poultry influenza reference laboratory.
Prepared by embodiment 2 bird flu H9 subtype avian influenza virus strain HD strain inactivated vaccine
1, the preparation of vaccine antigen liquid and malicious valency measure
By HD strain virus culture fluid PBS (pH7.3, the phosphate buffer of 0.01M) according to after 1:5000 ~ 1:10000 doubly (volume ratio) dilution, the nonimmune Embryo Gallus domesticus of inoculation 9-10 age in days, cultivate 96 hours (discarding dead Embryo Gallus domesticus in 24h) for 37 DEG C, after 4 DEG C of cold preservation 24h, collect allantoic fluid, carry out purification by the mode of continuous flow centrifugation afterwards, collect supernatant, the malicious valency measuring supernatant is generally: 10
8-10
9eLD
50, HA tires in 1:256 ~ 2, and 048.Described supernatant is for subsequent use in-20 DEG C of preservations as viral solution (antigen liquid)
2, antigen liquid deactivation
Add in viral solution by propiolactone solution according to certain ratio, deactivation 24 hours of vibrating under 2 DEG C ~ 8 DEG C conditions, gets the virus-culturing fluid after deactivation, through allantocherion vaccination 9-11 age in days SPF Embryo Gallus domesticus, and blind passage 3 generation.Carried out ten groups of inactivation experiments altogether, deactivation condition used in six groups is as table 1.Propiolactone solution final concentration used is for being respectively 1:1000,1:2000,1:3000,1:4000,1:5000, and through inspection, ten groups of inactivation of viruses, all without hemagglutination activity, are qualified inactivation of viruses solution.By inactivation of viruses solution after the assay was approved for the preparation of vaccine.According to the restriction of various condition and the principle of saving, it is 1:2000-1:4000 that deactivation condition is tentatively defined as propiolactone final concentration, and inactivation time is 20-30 hour.Virus liquid (antigen liquid) through above-mentioned condition inactivation treatment is for subsequent use in-20 DEG C of preservations
Table 1: propiolactone deactivation condition
3, oil phase adjuvant preparation
Special for medical grade white oil (MARCAL-52, Mobil), Si Ben-80 and aluminium stearate are pressed the proportions mixing of 94:6:2, for subsequent use after high temperature sterilize.
4, aqueous phase preparation
By a certain amount of tween 80 after high temperature sterilize, join in six groups of antigen liquids of deactivation, tween 80 final concentration is 3% (volume ratio), fully dissolves mixing be aqueous phase through spending the night.
5, vaccine formulation
By a certain proportion of oil phase and described six groups of aqueous phases (respectively with the ratio of 2:3 and 2:4, make six batches of vaccines) in vertical colloid mill machine after premix, mixture is injected into high-pressure homogenization pump internal emulsification again, make water in oil emulsion, in six groups of vaccines, every milliliter is all more than or equal to 10 containing virus quantity
8eID
50.
6, safety testing
Get SPF chicken 120 in 2 week age, 10/batches, often organize two batches, be divided into 12 groups, often criticize vaccine immunity and inject 2 groups, often organizing six batches of inactivated vaccines described in the cervical region subcutaneous injection of chicken with 3 times of immunizing doses (1.5mL) respectively.Establish matched group chicken 10 in addition, continuous 21d simultaneously, observe and whether occur inoculating by H9 subtype avian influenza (HD strain) deactivation oil seepage any local or systemic adverse reactions that cause.Result proves that all inoculation chickens all do not have limping symptom to occur, without other any clinical pathological symptoms or pathological change, without any local or general reaction yet.
Embodiment 3HD strain inactivated vaccine immune protective is tested
Get 25 age in days SPF chicken 130, wherein 120 according to 10/batch, often organize two batches, be divided into 12 groups, as immune group, often criticize vaccine immunity and inject 2 groups, other 10 as a control group.Immune group inoculates six groups of above prepared bird flu virus H9 hypotype inactivated vaccines respectively, and every intramuscular injection single dose vaccine 0.5ml, after 28 days, does challenge test with bird flu virus H9 hypotype HD strain.Remaining is matched group, a counteracting toxic substances not vaccination.Counteracting toxic substances dosage is 10
5.
0eID
50.Within after counteracting toxic substances 5 days, gather cotton swab, inoculation 9-10 age in days SPF Embryo Gallus domesticus is separated this virus.
Known by experiment, all there is not the clinical symptoms of doubtful bird flu in immune group, and cotton swab virus purification result is: the quantity that each immune group 20 is only separated to virus is 0, and the quantity that matched group 10 is only separated to virus is 10.Cotton swab result display bird flu virus H9 hypotype HD strain inactivated vaccine can make chicken from the attack of this strain, shows this strain and has good immune protective effect.
Took a blood sample after after a 0.5ml intramuscular injection 14,21,28 and 35 days, separation of serum measures its titre to H9 hypotype AIV.After 14 days, most of chicken has shown HI antibody in various degree.After 21 days, all immune chickens all produce the HI antibody of anti-H9, and significantly rise during compared with 14 days, and HI antibody titer peaked 35 days time, on average can reach 9.5log2.
Embodiment 4:HD strain Immunization cross-protection test
Get commercial bird flu H9 hypotype inactivated vaccine X strain and carry out Cross immunogenicity test with the inactivated vaccine that the bird flu H9 hypotype HD strain of the embodiment of the present invention 1 makes, the Cross immunogenicity performance of checking HD strain inactivated vaccine.
Get 25 age in days SPF chicken 120, be divided into 6 groups, often organize is all 20,1st group and the 2nd group of muscle and subcutaneous vaccination X strain inactivated vaccine every 0.5ml/ are only, only, the 3rd group and the 6th group of muscle and the aseptic PBS 0.5ml/ of subcutaneous vaccination are only for 4th group and the 5th group of muscle and subcutaneous vaccination HD strain inactivated vaccine 0.5ml/.In immunity latter 28 days, the 1st, 2,3 group with bird flu H9N2 hypotype HD strain counteracting toxic substances 10
5.5eID
50/ only, the 4th, 5,6 group with bird flu H9N2 hypotype X strain counteracting toxic substances 10
5.5eID
50/ only; After counteracting toxic substances, every day observes chicken public sentiment condition, observes two weeks, and gathers oral cavity and cloacal swabs at the 5th day, carries out virus purification etc.After counteracting toxic substances the 14th day, cut open all test chickens of inspection, observe viscera etc.
Result of the test shows (result of the test is in table 2).After counteracting toxic substances, so all there is not obvious clinical symptoms in group; And the display of cotton swab virus purification result, the 1st group has 5 parts of cotton swabs can be separated to AIV H9 subtype avian influenza virus, and the 3rd group and the 6th group of inoculation PBS almost all can be separated to virus.Illustrate that Avian Influenza Virus H9N2 HD strain can provide good protection for chicken, prevent the attack of HD strain and X strain; The antibody that Avian Influenza Virus H9N2 X strain produces can prevent the attack of X strain, and can only produce the immune protective efficiency of 75% to the attack of HD strain.Further illustrate bird flu H9N2 subtype virus HD strain inactivated vaccine of the present invention and have good cross-protection, can resist the attack of HD strain and other avian influenza strains, this further illustrates HD strain and has higher spectrum.
The SPF chicken immune counteracting toxic substances cross protection of the HD strain of table 2 H9 subtype avian influenza and X strain is tested
It should be noted that, in this article, term " comprises ", " comprising " or its any other variant are intended to contain comprising of nonexcludability, thus make to comprise the equipment of a series of key element or device not only comprises those key elements, but also comprise other key elements clearly do not listed, or also comprise this equipment or the intrinsic key element of device.
It is last it is noted that above embodiment is only in order to illustrate technical scheme of the present invention but not to be limited, although with reference to preferred embodiment to invention has been detailed description, those of ordinary skill in the art is to be understood that: it still can be modified to technical scheme of the present invention or equivalent replacement, and these are revised or be equal to the spirit and scope that replacement also can not make amended technical scheme disengaging technical solution of the present invention.
Claims (10)
1. a H9 subtype avian influenza virus, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), and preservation date is on October 22nd, 2014, and deposit number is CGMCC No.9813.
2. a H9 subtype avian influenza inactivated vaccine, is characterized in that, described vaccine comprises the H9 subtype avian influenza virus as claimed in claim 1 of deactivation.
3. a preparation method for H9 subtype avian influenza inactivated vaccine, is characterized in that, comprises the following steps:
A () carries out deactivation to the culture fluid comprising H9 subtype avian influenza virus according to claim 1, to obtain the solution containing deactivation H9 subtype avian influenza virus;
B () adds emulsifying agent to the described solution containing deactivation H9 subtype avian influenza virus and prepares aqueous phase; And
C described aqueous phase and oil phase are carried out mixing and emulsifying by (), to obtain described H9 subtype avian influenza inactivated vaccine.
4. method as claimed in claim 3, it is characterized in that, before step (a), described method also comprises to be inoculated in nonimmune Embryo Gallus domesticus by H9 subtype avian influenza virus as claimed in claim 1, and cultivate at 35.5 ~ 37.5 DEG C, the chick embryo allantoic liquid that collection obtains also carries out centrifugal purification by the mode of continuous flow centrifugation, collects supernatant to obtain the culture fluid comprising H9 subtype avian influenza virus described in claim 1.
5. the method as described in claim 3 or 4, it is characterized in that, under deactivation described in step (a) is included in 2 ~ 8 DEG C of conditions, propiolactone (propiolactone: the culture fluid) culture fluid to the described H9 of comprising subtype avian influenza virus utilizing final concentration to be 1:2000 ~ 1:4000 carries out inactivation treatment 16 ~ 36 hours; And propiolactone was hydrolyzed in 2 hours 37 DEG C of Water Under solutions, to obtain the solution containing deactivation H9 subtype avian influenza virus.
6. the method as described in claim 3 or 4, it is characterized in that, aqueous phase of preparing described in step (b) comprises and mixes, the described solution containing deactivation H9 subtype avian influenza virus to obtain aqueous phase with the volume ratio of tween 80 according to 94 ~ 97:3 ~ 6; Wherein, the concentration of described tween 80 is 3.0%.
7. the method as described in claim 3 or 4, is characterized in that, the preparation of described oil phase comprises and mixes, medical grade white oil (MARCAL-52, Mobil), Si Ben-80 and aluminium stearate to obtain described oil phase according to the mass ratio of 94:6:2.
8. the method as described in claim 3 or 4, it is characterized in that, in step (c), described aqueous phase and oil phase are carried out mixing and emulsifying to comprise and described aqueous phase and oil phase are carried out mixing and emulsifying according to the ratio of volume ratio 2:3 ~ 2:4, to obtain described H9 hypotype disease vaccine.
9. the application of H9 subtype avian influenza virus in the preparation of the medicine of prevention and therapy H9 subtype avian influenza as claimed in claim 1.
10. the application of H9 subtype avian influenza inactivated vaccine in the preparation of the medicine of prevention and therapy H9 subtype avian influenza as claimed in claim 2.
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| CN106924727A (en) * | 2017-04-26 | 2017-07-07 | 广州博恒生物科技有限公司 | A kind of preparation method of avian influenza virus H9 hypotype inactivated vaccines |
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| CN111718909A (en) * | 2020-06-30 | 2020-09-29 | 肇庆大华农生物药品有限公司 | Method for inactivating virus in production of avian influenza H9 subtype vaccine |
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| CN106924727B (en) * | 2017-04-26 | 2020-06-23 | 广州渔跃生物技术有限公司 | Preparation method of avian influenza virus H9 subtype inactivated vaccine |
| CN109091670A (en) * | 2018-08-22 | 2018-12-28 | 广州市华南农大生物药品有限公司 | A kind of avian influenza virus H9 hypotype bivalent inactivated vaccine and preparation method thereof |
| CN109091670B (en) * | 2018-08-22 | 2020-08-28 | 广州市华南农大生物药品有限公司 | Avian influenza virus H9 subtype bivalent inactivated vaccine and preparation method thereof |
| CN111718909A (en) * | 2020-06-30 | 2020-09-29 | 肇庆大华农生物药品有限公司 | Method for inactivating virus in production of avian influenza H9 subtype vaccine |
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Application publication date: 20150429 |