CN103396999A - H9N2 subtype virus, H9N2 subtype virus vaccine and preparation method thereof - Google Patents
H9N2 subtype virus, H9N2 subtype virus vaccine and preparation method thereof Download PDFInfo
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Abstract
The invention discloses an H9N2 subtype virus, an H9N2 subtype virus vaccine and a preparation method thereof. Specifically, the H9N2 subtype virus is preserved in China General Microbiological Culture Collection Center with a preservation number of CGMCC:7702. The H9N2 subtype virus has strong virulence, good immunogenicity, and high toxic valence. The vaccine produced by the H9N2 subtype virus has good safety and long immune protection period.
Description
Technical field
The present invention relates to biological technical field, be specifically related to H9N2 subtype virus and H9N2 hypotype disease vaccine and preparation method thereof.
Background technology
Bird flu (Avian influenza, AI) is the hyperinfection disease of the serious harm aviculture that caused by A type avian influenza virus (Avian influenza virus, AIV).Avian influenza virus belongs to orthomyxoviridae family, Influenza Virus.Hemagglutinin (Hemagglutinin, HA) and neuraminidase (Nueraminidase, NA) are two kinds of main protection antigen of virus,, according to the antigenic specificity of HA, NA, A type influenza virus can be divided into different hypotypes.Up to now, the HA of A type avian influenza virus has found 16 kinds, and NA has 9 kinds, respectively with H1～H16, N1～N9 name, no cross reaction between different H antigens or N antigen.Between each hypotype, the hemagglutinin amino acid sequence homology is below 70%, and identical hypotype amino acid sequence homology is 80%～90%.
This disease betided Italy first in 1878, later both at home and abroad many scholars have reported generation that should disease and the tremendous economic loss that causes to poultry husbandry thereof in succession.The H9N2 subtype avian influenza is the avian influenza virus subtype the most widely that distributes in the world at present.The H9N2 subtype avian influenza virus separated in the body of the turkey from North America early than 1966 obtain, and the influenza virus of this hypotype only infects turkey, infected chicken hardly at first.But since the nineties in 20th century, just a lot of poulty houses spread the H9N2 subtype avian influenza virus in Asia.This disease has been popular in many countries and regions in the world at present, to aviculture, has caused huge financial loss.In China, high pathogenic avian influenza is take the H5N1 hypotype as representative, and the low pathogenicity bird flu is take the H9N2 hypotype as principal mode.
The AIV blood serum subtype is more, and different subtype AIV is even not different to the susceptibility of different hosts with strain, and susceptibility changes and can change because of virus variation.HA genovariation rate is high, is the major cause that antigenic variation occurs virus.Maturation and release that NA albumen is main and viral have substantial connection, may have influence on the copying of virus, propagation etc.Because the avian influenza virus variation is very fast, bring difficulty to safety control of bird flu, the R﹠D cycle of curative drug is longer, has affected to a certain extent clinical control effect.Vaccination is the Main Means of prevention and control of fowl influenza, but because the avian influenza virus variation is fast, epidemic isolates is many, and serology is many and without cross immunity, its effect is restricted greatly.Solve this difficult problem, depend on the epidemic characteristic to the popular strain in this area, the variation characteristics are studied and screening and separating goes out suitable advantage vaccine strain, so that the prevention and control of bird flu have more specific aim and validity.
Summary of the invention
The present invention one of is intended to solve the problems of the technologies described above at least to a certain extent or provides at least a kind of useful business to select.For this reason, the object of the invention is to propose a kind of H9N2 subtype virus and H9N2 hypotype disease vaccine and preparation method thereof.
In a first aspect of the present invention, the present invention proposes a kind of H9N2 subtype virus, according to the embodiment of the present invention, this H9N2 subtype virus deposit number CGMCC:7702, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, according to the embodiment of the present invention, described H9N2 subtype virus is to separate and obtain from the morbidity chicken group through normal immune inactivated avian influenza vaccine, its malicious valency EID
50Be 10
7.5～10
8.5/ ml, therefore, described H9N2 subtype virus strain toxicity is good, has good immunogenicity, can be used as inactivated vaccine and produces strain and check seed culture of viruses.
In a second aspect of the present invention; the present invention proposes the purposes of above-mentioned H9N2 subtype virus in preparation H9N2 hypotype disease vaccine; in one embodiment of the invention; protection test proves according to H9N2 subtype virus cross immunity, and described H9N2 subtype virus has good cross immunity provide protection.Therefore utilize this H9N2 subtype virus to produce H9N2 hypotype disease vaccine, can effectively improve the immunogenicity of H9N2 hypotype disease vaccine, thereby further improve the disease vaccine security of H9N2 hypotype and immune period.
In third aspect present invention, the present invention proposes a kind of H9N2 hypotype disease vaccine, according to the embodiment of the present invention, this H9N2 hypotype disease vaccine comprises the H9N2 subtype virus of deactivation.Thereby effectively improve the immunogenicity of H9N2 hypotype disease vaccine, further improve the disease vaccine security of H9N2 hypotype and immune period.
According to the embodiment of the present invention, the H9N2 hypotype disease vaccine of the above embodiment of the present invention also comprises following additional technical feature:
According to one embodiment of present invention, described H9N2 hypotype disease vaccine, by formalin-inactivated, can effectively improve the immunogenicity of H9N2 hypotype disease vaccine thus, can further improve the disease vaccine security of H9N2 hypotype and immune period simultaneously.
In fourth aspect present invention, the present invention proposes a kind of method of the H9N2 of preparation hypotype disease vaccine, according to the embodiment of the present invention, the method comprises: H9N2 subtype virus claimed in claim 1 is carried out deactivation, in order to obtain the H9N2 subtype virus through deactivation; Utilize described H9N2 subtype virus through deactivation to prepare water; And described water and oil phase are carried out mixing and emulsifying, in order to obtain described H9N2 hypotype disease vaccine.Thus, the immunogenicity of H9N2 hypotype disease vaccine be can effectively improve, thereby the disease vaccine security of H9N2 hypotype and immune period further improved.
According to one embodiment of the invention, the method for preparing H9N2 hypotype disease vaccine of the above embodiment of the present invention also comprises following additional technical feature:
According to the embodiment of the present invention, before described H9N2 subtype virus is carried out deactivation, comprise according to the following step and obtain H9N2 subtype virus nutrient solution: in the nonimmune chicken embryo of described H9N2 subtype virus inoculation, and cultivate under 35.5～37.5 ℃, collect the chick embryo allantoic liquid that obtains; Separate described chick embryo allantoic liquid, in order to obtain described H9N2 subtype virus nutrient solution.
, according to one embodiment of the invention, described H9N2 subtype virus is carried out deactivation comprise: under 37 ℃, utilizing final concentration is that 0.2% formaldehyde carried out inactivation treatment 16～36 hours to H9N2 subtype virus nutrient solution; In order to obtain to contain the solution of deactivation H9N2 subtype virus.According to one embodiment of the invention, the described water for preparing comprises: the solution that will contain deactivation H9N2 subtype virus mixes with the volume ratio of tween-80 according to 96:4, in order to obtain described water.、
According to one embodiment of the invention, the described oil phase for preparing comprises: injection white oil MARCOL52, Si Ben-80 and the aluminum stearate mass ratio according to 94:6:2 is mixed, in order to obtain described oil phase.Thus, improve the immunogenicity of H9N2 hypotype disease vaccine, thereby further improve the disease vaccine security of H9N2 hypotype and immune period.
According to one embodiment of the invention, described water is mixed and comprises with oil phase: be that 1:2～2:3 carries out the mixing and emulsifying stirring with described water and oil phase according to volume ratio, adding final concentration before stirring termination is 0.01% Thiomersalate in order to obtain described H9N2 hypotype disease vaccine.Thus, improve the immunogenicity of H9N2 hypotype disease vaccine, thereby further improve the disease vaccine security of H9N2 hypotype and immune period.
Additional aspect of the present invention and advantage part in the following description provide, and part will become obviously from the following description, or by practice of the present invention, recognize.
Description of drawings
Above-mentioned and/or additional aspect of the present invention and advantage are from obviously and easily understanding becoming the description of embodiment in conjunction with following accompanying drawing, wherein:
Fig. 1 is preparation method's Technology Roadmap of H9N2 hypotype disease vaccine according to an embodiment of the invention.
Embodiment
The present invention is based on contriver's following discovery and completes: the contriver has separated a strain H9N2 subtype virus, and this virus can, as preparation H9N2 hypotype disease vaccine, be used for preventing bird flu after deactivation.
In a first aspect of the present invention, the present invention proposes a kind of H9N2 subtype virus, according to the embodiment of the present invention, this H9N2 subtype virus deposit number CGMCC:7702, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, according to the embodiment of the present invention, the H9N2 subtype virus is to separate and obtain from the morbidity chicken group through normal inactivated avian influenza vaccine immunity, its malicious valency EID
50Be 10
7.5～10
8.5/ ml; therefore; separate the H9N2 subtype virus strain toxicity that obtains good; has good immunogenicity; can be used as inactivated vaccine and produce strain and check seed culture of viruses; according to one embodiment of the invention, the protection test of H9N2 subtype virus cross immunity proves, this H9N2 subtype virus has good cross immunity provide protection.Therefore can utilize this H9N2 subtype virus to produce H9N2 hypotype disease vaccine, effectively improve the immunogenicity of H9N2 hypotype disease vaccine, thereby further improve the disease vaccine security of H9N2 hypotype and immune period.
In a second aspect of the present invention; the present invention proposes the purposes of above-mentioned H9N2 subtype virus in preparation H9N2 hypotype disease vaccine; in one embodiment of the invention, protection test proves according to H9N2 subtype virus cross immunity, and this H9N2 subtype virus has good cross immunity provide protection.Therefore utilize this H9N2 subtype virus to produce H9N2 hypotype disease vaccine, can effectively improve the immunogenicity of H9N2 hypotype disease vaccine, thereby further improve H9N2 hypotype disease vaccine immune period and immune effect.
In a third aspect of the present invention, the present invention proposes a kind of H9N2 hypotype disease vaccine, according to the embodiment of the present invention, this H9N2 hypotype disease vaccine comprises the H9N2 subtype virus of deactivation.According to the embodiment of the present invention, the immune duration of this H9N2 hypotype disease vaccine can be kept 4 months at least.According to the embodiment of the present invention, this H9N2 hypotype disease vaccine can be preserved 12 months under 2～8 degrees centigrade, so this vaccine has satisfactory stability.
According to the embodiment of the present invention, above-mentioned H9N2 hypotype disease vaccine is inactivated, the inactivator kind that can adopt also is not particularly limited, as long as can effectively destroy the nucleic acid construct of H9N2 subtype virus and make protein denaturation, but does not destroy antigenicity and the blood clotting of H9N2 subtype virus.According to one embodiment of the invention, the inactivator that adopts is formaldehyde, and formaldehyde can effectively destroy the DNA structure of H9N2 subtype virus, and then reaches the purpose of deactivation H9N2 subtype virus.According to the embodiment of the present invention, the inactivator formaldehyde final concentration that adopts also is not particularly limited, as long as effective deactivation H9N2 subtype virus.According to one embodiment of the invention, the final concentration of the formaldehyde that adopts is 0.2%.
In a fourth aspect of the present invention, the present invention proposes a kind of method of the H9N2 of preparation hypotype disease vaccine, according to the embodiment of the present invention, the method comprises carries out deactivation to above-mentioned H9N2 subtype virus, in order to obtain the H9N2 subtype virus through deactivation; Utilize the H9N2 subtype virus through deactivation to prepare water; And water and oil phase are carried out mixing and emulsifying, in order to obtain H9N2 hypotype disease vaccine.Thus, effectively improve the immunogenicity of H9N2 hypotype disease vaccine, thereby further improve H9N2 hypotype disease vaccine immune period and immune effect.
According to one embodiment of present invention, before the H9N2 subtype virus is carried out deactivation, comprise according to the following step and obtain H9N2 subtype virus nutrient solution: the H9N2 subtype virus is inoculated in nonimmune chicken embryo, and cultivate under 35.5～37.5 ℃, collect the chick embryo allantoic liquid that obtains, in order to obtain H9N2 subtype virus nutrient solution.According to the embodiment of the present invention, nonimmune chicken embryo is instar chicken embryo on the 9th～11, can effectively cultivate the H9N2 subtype virus., according to one embodiment of the invention, before the H9N2 subtype virus is inoculated in nonimmune chicken embryo, first carry out the H9N2 subtype virus rare, after inoculation, cultivated under 35.5～37.5 ℃ 72 hours, and discarded dead chicken embryo in 24 hours, collect 24～72 hours dead chicken embryos.Thereby be conducive to obtain the high chick embryo allantoic liquid of malicious valency, and then improve the immune protective efficiency of preparation H9N2 hypotype disease vaccine.
According to one embodiment of the invention, the H9N2 subtype virus is carried out the condition of deactivation and is not particularly limited, according to a particular embodiment of the invention, can adopt formaldehyde to carry out deactivation to the H9N2 subtype virus.The inactivator formaldehyde final concentration that adopts also is not particularly limited, as long as effective deactivation H9N2 subtype virus.The final concentration of the formaldehyde that adopts according to a particular embodiment of the invention, is 0.1%～0.4%.According to the embodiment of the present invention, the time of inactivator deactivation and temperature also are not particularly limited, as long as can effectively destroy the nucleic acid construct of H9N2 subtype virus.According to a particular embodiment of the invention, utilizing final concentration is that 0.1%～0.4% formaldehyde carried out inactivation treatment 16～36 hours to H9N2 subtype virus nutrient solution, thus can be with the abundant deactivation of H9N2 subtype virus.
According to one embodiment of present invention, in the above-mentioned method for preparing H9N2 hypotype disease vaccine, can utilize following method to prepare water: the solution that will contain deactivation H9N2 subtype virus mixes with tween-80.
According to a particular embodiment of the invention, contain the solution of deactivation H9N2 subtype virus and proportioning that tween-80 mixes and be not particularly limited,, according to concrete example of the present invention, can mix according to the volume ratio of 96:4.By adopting said ratio and concentration to prepare water.
According to one embodiment of present invention, in the above-mentioned method for preparing H9N2 hypotype disease vaccine, can utilize following method to prepare oil phase: injection white oil MARCOL52, Si Ben-80 and aluminum stearate to be mixed, in order to obtain oil phase.According to a particular embodiment of the invention; the quality that injection white oil MARCOL52, Si Ben-80 and aluminum stearate can adopt is 94:6:2; the immunogenicity of H9N2 hypotype disease vaccine be can improve thus, thereby H9N2 hypotype disease vaccine immune period and immune protective effect further improved.
According to one embodiment of present invention,, with water and oil phase mixing and emulsifying, can be that 1:2～2:3 carries out the mixing and emulsifying stirring according to water and oil phase volume ratio, add 0.01 Thiomersalate in order to obtain H9N2 hypotype disease vaccine before stopping stirring.Thus can be so that suitably degree emulsification improve the immunogenicity of H9N2 hypotype disease vaccine, thus the immune protective effect of H9N2 hypotype disease vaccine further improved.
Below with reference to specific embodiment, present invention is described, need to prove, these embodiment are only descriptive, and do not limit the present invention in any way.
Separation and the evaluation of embodiment 1 bird flu H9N2 subtype virus strain Jiangsu strain
1, virus is separated
Gather disease fowl sample and comprise tracheae, lung, cloaca swab, collected specimens is placed in the isotonic phosphate buffer liquid (PBS) of the pH value 7.0～7.4 that contains antibiotic.The cotton swab that is accredited as positive is fully twisted, discard swab after wringing out, sample liquid, through the centrifugal 10min of 1500r/min, is got supernatant liquor and is filtered with 0.22 μ m sterilization filter, the sterile tissue sample of handling well is inoculated 9～11 age in days SPF chicken embryos through the fine hair allantoic cavity, inoculate altogether 10 pieces, 0.2mL/ piece, put 37 ℃ and hatch, every 8h is according to an embryo, discard non-specific dead embryo in 24h, collect the dead embryo allantoic liquid of 24～72h, place-20 ℃ of Cryopreservations standby.
According to the method described above, be separated to the virus of blood clotting from the pathological material of disease of sick chicken, HA evidence Jiangsu strain can the aggegation chicken red blood cell, the blood clotting of HI evidence Jiangsu strain can not be suppressed by Avian pneumo-encephalitis virus (NDV) antiserum(antisera), the cleer and peaceful anti-AIV H7 hypotype serum of anti-AIV H5 subgroup blood, can be suppressed by AIV H9 hypotype positive serum.The results are shown in Table 1.
The first separating resulting of table 1 pathological material of disease
2, hypotype is identified
The Jiangsu strain chick embryo allantoic liquid that separates is accredited as AIV H9N2 hypotype through hypotype, and called after A/Chicken/Jiangsu-12/2012 (H9N2 hypotype) strain, be called for short the Jiangsu 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 May 29th, 2013, and Classification And Nomenclature is influenza A virus, and preserving number is CGMCC No.7702.Identify with serum available from country of Harbin Veterinary Medicine Inst., China Academy of Agriculture bird flu reference laboratory.
Embodiment 2 bird flu H9N2 subtype virus strain Jiangsu strain inactivated vaccine preparations
1, the preparation of vaccine antigen liquid and malicious valency are measured
With the Jiangsu strain virus with normal saline dilution after, the inoculation 10 nonimmune chicken embryos of age in days, cultivate 72 hours (discarding dead chicken embryo in 24h) for 37 ℃, collects and killed by a pinch the chick embryo allantoic liquid of dying in 24-72 hour, measures malicious valency: 10
7.5-10
8.5EID
50It is standby that virus liquid is put-20 ℃ of preservations
2, antigen liquid deactivation
Formaldehyde solution is added in virus liquid, and final concentration is 0.2%, puts in 37 ℃ of incubators the effect of shaking 20 hours, through inoculating 9～11 age in days SPF chicken embryos after the assay was approved as preparing antigen liquid.
3, oil phase adjuvant preparation
With the 94:6:2 ratio preparation mixing in mass ratio of the special-purpose white oil of injection, Si Ben-80 and aluminum stearate, standby after high-temperature sterilization.
4, water preparation
The tween-80 of 4 volumes after high-temperature sterilization, is joined in the liquid of inactivation antigen of 96 volumes, through fully dissolving to mix, be water.
5, vaccine preparation
A certain proportion of oil phase and water (ratio is 1:2) in the vertical colloid mill machine after premix, then are injected into high-pressure homogenization pump internal emulsification with mixture, make water-in-oil emulsion, every milliliter contains virus quantity more than or equal to 1.5 * 10
8.0EID
50
6, safety testing
Get 2 age in week 10 of SPF chickens, 3 times of immunizing dose inactivated vaccines of every chicken neck subcutaneous injection.Establish simultaneously 10 of control group chickens, 14d, observe and any part or the systemic adverse reactions that are caused by the inoculation of bird flu H9N2 hypotype Jiangsu strain deactivation oil seepage whether occur continuously.Result proves that all inoculation chickens all do not have the limping symptom to occur, also without other any clinical pathology symptom or pathological change, without any part or systemic reaction.
Embodiment 3 Jiangsu strain inactivated vaccine immune protective tests
Get 20 of 28 age in days SPF chickens, be divided into 2 groups, 10 every group, be divided into immune group and control group.Immune group inoculation Avian Influenza Virus H9N2 Jiangsu strain inactivated vaccine, every intramuscular injection single dose vaccine 0.5ml, after 28 days, do challenge test with the strain of Avian Influenza Virus H9N2 Jiangsu.Remaining is control group, only attacks not vaccination of poison.Attack the toxic agent amount and be 10
5.0EID
50Attack poison and gathered cotton swab in rear 5 days, inoculate instar chicken embryo on the 10th and separate this virus.
The clinical symptom of doubtful bird flu does not all appear in (test-results is in Table 6) by experiment as can be known, immune group, and cotton swab virus separating resulting is: the quantity that 10 of immune group are separated to virus is 0, and the quantity that 10 of control groups are separated to virus is 10.The cotton swab result shows that strain inactivated vaccine in Avian Influenza Virus H9N2 Jiangsu can make chicken avoid the attack of this strain, shows that this strain has good immune protective effect.
Table 6SPF chicken morbidity and cotton swab virus separating resulting
With blood sampling afterwards in 14,21,28 and 35 days after an intramuscular injection of 0.5ml, separation of serum is measured its titre to H9N2 hypotype AIV.After 14 days, most of chickens have shown HI antibody in various degree.After 21 days, all immune chickens all produce the HI antibody of anti-H9, and significantly rise during than 14 days, and the HI antibody titers peaked in the time of 35 days, on average can reach 10log2.
Embodiment 4 Jiangsu strain Immunization cross-protection tests
Get commercial bird flu H9 hypotype inactivated vaccine H strain and with the inactivated vaccine that the bird flu H9 hypotype Jiangsu strain of the embodiment of the present invention 1 is made, carry out the cross immunity protection test, the immune protection performance of checking Jiangsu strain inactivated vaccine.
Get 50 of 28 age in days SPF chickens, be divided into 6 groups, the 1st group, the 2nd group, the 4th group and the 5th group is all 10, the 3rd group and the 6th group is all 5, the 1st group and the 2nd group of muscle and every 0.5ml/ of subcutaneous vaccination H strain inactivated vaccine are only, only, the 3rd group and the 6th group of muscle and the aseptic PBS0.5ml/ of subcutaneous vaccination are only for the 4th group and the 5th group of muscle and subcutaneous vaccination Jiangsu strain inactivated vaccine 0.5ml/.In immunity rear 28 days, attack poison 10 with the Jiangsu strain of bird flu H9N2 hypotype for the 1st, 2,3 group
5EID
50/ only, attack poison 10 with bird flu H9N2 hypotype H strain for the 4th, 5,6 group
5EID
50/ only; After attacking poison, observe the chicken public sentiment condition every day, observed for two weeks, and at the 5th day, gather oral cavity and cloacal swabs, carries out that virus is separated etc.Attack poison rear the 14th day, and cutd open all test chickens of inspection, observe viscera etc.
Test-results shows (test-results is in Table 7).After attacking poison, so obvious clinical symptom does not all appear in group; And cotton swab virus separating resulting shows, the 1st group has 2 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 all can be separated to virus.Illustrate that the strain of Avian Influenza Virus H9N2 Jiangsu can provide good protection for chicken, prevents the attack of Jiangsu strain and H strain; The antibody that Avian Influenza Virus H9N2 H strain produces can prevent the attack of H strain, and to the attack of Jiangsu strain, can only produce 80% immune protective efficiency.Further illustrate bird flu H9N2 subtype virus Jiangsu strain inactivated vaccine of the present invention and have cross-protection preferably, can resist the attack of Jiangsu strain and other avian influenza strains.
The SPF chicken immune of table 7H9 subtype avian influenza Jiangsu strain and H strain is attacked malicious cross protection experiment
Preparation and the stability test of embodiment 5 Jiangsu strain stable antigens
With avian influenza virus Jiangsu strain inoculation 10 age in days SPF chicken embryos, discard the 24h dead germ, the allantoic fluid of results 24h-96h dead germ, allantoic fluid is carried out deactivation, after centrifugal, add proper adjuvant to make the stable antigen of avian influenza virus Jiangsu strain, there is not the danger of loose poison in this antigen when using and lay in, reduced because use live virus antigen the harm of environment and human body, this product has good stability, under 2～8 ℃ of conditions, preserve more than 12 months this product blood clotting valency constant, the HI that can be used for bird flu H9N2 hypotype detects (the results are shown in Table 8).
Table 8 Jiangsu strain stable antigen storage stability test-results
In the description of this specification sheets, the description of reference term " embodiment ", " some embodiment ", " example ", " concrete example " or " some examples " etc. means to be contained at least one embodiment of the present invention or example in conjunction with specific features, structure, material or the characteristics of this embodiment or example description.In this manual, the schematic statement of above-mentioned term not necessarily referred to identical embodiment or example.And the specific features of description, structure, material or characteristics can be with suitable mode combinations in any one or more embodiment or example.
Although the above has illustrated and has described embodiments of the invention, be understandable that, above-described embodiment is exemplary, can not be interpreted as limitation of the present invention, those of ordinary skill in the art is not in the situation that break away from principle of the present invention and aim can change above-described embodiment within the scope of the invention, modification, replacement and modification.
Claims (10)
1. a H9N2 subtype virus (A/Chicken/Jiangsu-12/2012 (H9N2 hypotype) strain is called for short the Jiangsu strain), preserving number CGMCC:7702, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center.
2. the purposes of H9N2 subtype virus claimed in claim 1 in preparation H9N2 hypotype disease vaccine.
3. a H9N2 hypotype disease vaccine, is characterized in that, comprises:
The H9N2 subtype virus claimed in claim 1 of deactivation.
4. H9N2 hypotype disease vaccine according to claim 3, is characterized in that, described H9N2 subtype virus is by formalin-inactivated.
5. a method for preparing H9N2 hypotype disease vaccine, is characterized in that, comprising:
H9N2 subtype virus claimed in claim 1 is carried out deactivation, in order to obtain the H9N2 subtype virus through deactivation;
Utilize described H9N2 subtype virus through deactivation to prepare water; And
Described water and oil phase are carried out mixing and emulsifying, in order to obtain described H9N2 hypotype disease vaccine.
6. method according to claim 5, is characterized in that, before described H9N2 subtype virus is carried out deactivation, comprises according to the following step and obtain H9N2 subtype virus nutrient solution:
Described H9N2 subtype virus is inoculated in nonimmune chicken embryo, and cultivates under 35.5～37.5 degrees centigrade;
Collect the chick embryo allantoic liquid that obtains, in order to obtain described H9N2 subtype virus antigen.
7. method according to claim 5, is characterized in that, described H9N2 subtype virus carried out deactivation comprise:
Under 37 degrees centigrade, utilizing final concentration is that 0.1%～0.4% formaldehyde carried out inactivation treatment 16～36 hours to H9N2 subtype virus nutrient solution.
8. method according to claim 5, is characterized in that, the described water for preparing comprises:
The solution that will contain deactivation H9N2 subtype virus mixes with the volume ratio of tween-80 according to 96:4, in order to obtain mixed solution.
9. method according to claim 5, is characterized in that, the described oil phase for preparing comprises:
Injection white oil MARCOL52, Si Ben-80 and the aluminum stearate mass ratio according to 94:6:2 is mixed, in order to obtain described oil phase.
10. method according to claim 5, is characterized in that, described water is mixed and comprises with oil phase:
Be that 1:2～2:3 carries out mixing and emulsifying and stirs with described water and oil phase according to volume ratio, adding final concentration before stopping stirring is 0.01% Thiomersalate, in order to obtain described H9N2 subtype virus vaccine.
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| CN110951698A (en) * | 2019-12-25 | 2020-04-03 | 哈药集团生物疫苗有限公司 | Avian influenza virus TJ strain, avian influenza inactivated vaccine and preparation method thereof |
| CN111154732A (en) * | 2019-12-27 | 2020-05-15 | 天津渤海农牧产业联合研究院有限公司 | Avian influenza H9 subtype virus strain and application thereof |
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| CN102816740A (en) * | 2012-09-03 | 2012-12-12 | 江苏省农业科学院 | Avian influenza virus, inactivated vaccine and method for preparing same |
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| Publication number | Priority date | Publication date | Assignee | Title |
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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 |
| CN110951698A (en) * | 2019-12-25 | 2020-04-03 | 哈药集团生物疫苗有限公司 | Avian influenza virus TJ strain, avian influenza inactivated vaccine and preparation method thereof |
| CN111154732A (en) * | 2019-12-27 | 2020-05-15 | 天津渤海农牧产业联合研究院有限公司 | Avian influenza H9 subtype virus strain and application thereof |
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