CN103736088A - Avian influenza H9 subtype inactivated vaccine, preparation method and application thereof - Google Patents
Avian influenza H9 subtype inactivated vaccine, preparation method and application thereof Download PDFInfo
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Abstract
The present invention relates to an avian influenza H9 subtype inactivated vaccine, which is formed by mixing two strains of inactivated H9 subtype avian influenza viruses and an oil adjuvant, wherein the two strains of the inactivated H9 subtype avian influenza viruses are respectively the HZ strain and the FJ strain, the HA gene sequence of the HZ strain is represented by SEQ ID NO:1, and the HA gene sequence of the FJ strain is represented by SEQ ID NO:2. According to the preparation method, the virus solution of the HZ strain and the virus solution of the FJ strain of the H9 subtype avian influenza viruses are prepared and inactivated, the oil phase solution and the water phase solution are prepared, and emulsification is performed to obtain the finished product. According to the present invention, the two strains of the H9 subtype avian influenza viruses separated from different places are utilized to prepare the inactivated vaccine with characteristics of strong immunogenicity and good cross-protection property, wherein the inactivated vaccine can be used for prevention of chicken H9 subtype avian influenza diseases. In addition, after the inactivated vaccine is adopted to immunize chicken, the antibody titer is high so as to make the chicken have good virus challenge protection property on the H9 subtype strains epidemic in different places, the safety is high, and the efficacy is stable.
Description
Technical field
The invention belongs to animal biological product technical field, be specifically related to a kind of bird flu H9 hypotype inactivated vaccine and its preparation method and application.
Background technology
Bird flu (Avian Influenza, AI) is that the birds that caused by the A type influenza virus of Influenza Virus infects and the general name of disease.The poultry such as chicken, turkey, duck and Carnis Coturnicis japonicae and wild bird, aquatic bird, seabird etc. all can infect, and the harm that domestic chicken and turkey are caused is the most serious, it may be the worldwide popular main cause of bird flu that migratory bird, pet bird, loose fowl carry that virus migrates, and pig etc. also can be used as " blender " and propagate primary disease.
Difference according to bird flu virus AIV to Pathogenicity to fowl, can be divided into AIV high pathogenic avian influenza virus (HPAIV) and Lowly Pathogenic Avian Influenza Virus (LPAIV).H9 subtype avian influenza belongs to low pathogenicity influenza virus, it is the AIV Main Subtype existing in current China chicken group, although fatality rate not high (being generally no more than 30%) after this virus morbidity, but often cause respiratory symptom, the egg drop reduction of laying hen, and make the serious respiratory tract disease of the easy secondary of chicken group, affect performance of poultry.Its epidemic characteristic is at present: H9 hypotype is still the Main Subtype of current popular; H9 hypotype morphs; H9 hypotype become distribute or endemicity popular; H9 hypotype presents the pathogenic characteristic of conditionality; H9 hypotype is day by day serious to broiler chicken harm.
AIV hereditary variation is very frequent, and blood serum subtype is numerous.Research finds, AIV can by based on point mutation, gene recombinaton and sections reset the variation occurring in various degree, thereby make antigenicity and pathogenic the changing of influenza virus.The variation of AIV mainly occurs on HA and NA gene, especially HA gene, and its molecule mechanism mainly contains: the generation of antigenic drift, antigenic shift, RNA restructuring and defective virus granule etc.In recent years, along with the development of reverse genetics, bird flu hereditary variation and its mechanism were progressively confirmed.With regard to H5 hypotype, according to the variation situation of current H5 hypotype, adopt Reverse Genetics to obtain Rec-1, Rec-4, Rec-5 and Rec-6 vaccine strain, aspect the prevention and control of high pathogenic avian influenza, obtaining certain achievement.Variation research about H9 hypotype is also always underway.Liu Yan is studied the antigenic variation of the H9 subtype avian influenza virus separating between 1998-2002; through intersecting HI test, Embryo Gallus domesticus neutralization test, cell neutralization test, intersection protest test and HA gene analysis, proved that antigenic drift has occurred H9 subtype avian influenza virus.The people's such as Jiang Beiyu research shows; 1998-2008 has mostly produced good cross-protection between 4 strain H9 subtype avian influenza epidemic isolates of Beijing and Hebei province's separation, can protect the attack of epidemic isolates in 2008 with inactivated vaccine prepared by the epidemic isolates that 1998-2006 separates.Visible, whether to morph for H9 subtype avian influenza virus epidemic isolates antigenicity problem, there is larger difference in the conclusion (of pressure testing) that different reports draw.
Vaccine immunity is still one of current prevention and control H9 subtype avian influenza effective way.Along with the extensive use of H9 subtype avian influenza inactivated vaccine list Seedling or connection Seedling; in the last few years; under the effect of immune pressure; the vaccine strain that starts to use before occurring declines to the immune protective efficiency of terrain isolated strain; many cultivation units need the numerous and confused generation that adopts terrain epidemic isolates to make inactivated vaccine to come prevention and control H9 subtype avian influenza according to autoimmune; but these vaccines can not regular production, sale and spread, and can not guarantee its safety.Therefore,, for the present epidemic characteristic of epidemic disease, again screen safe, effective, stable bird flu H9 subtype virus strain and prepare H9 hypotype inactivated vaccine and seem very necessary.
Summary of the invention
In order to overcome the deficiencies in the prior art, the object of the present invention is to provide a kind of different local H9 subtype avian influenza strains that separate of 2 strains that utilize to be prepared into the inactivated vaccine that a kind of immunogenicity is strong, intersecting protective is good, for preventing chicken H9 subtype avian influenza.
For addressing the above problem, the technical solution adopted in the present invention is as follows:
A kind of bird flu H9 hypotype inactivated vaccine, is mixed and forms with oily adjuvant after deactivation by 2 strain H9 subtype avian influenza virus, and 2 strain H9 subtype avian influenza virus are respectively HZ strain and FJ strain, and its preservation registration number is respectively CGMCC NO.8355 and CGMCC NO.8354; The HA gene order of HZ strain is as shown in SEQ ID NO:1, and the HA gene order of FJ strain is as shown in SEQ ID NO:2.The center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms is submitted in HZ strain and FJ strain on October 15th, 2013, above-mentioned preservation strain is received at this center on October 18th, 2013, preservation date is on October 18th, 2013, this centre address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, postcode 100101.
A preparation method for bird flu H9 hypotype inactivated vaccine, is characterized in that comprising the steps:
1) prepare H9 subtype avian influenza virus HZ strain venom: H9 subtype avian influenza virus HZ strain normal saline is done to 10
-4dilution, inoculation 9～11 age in days SPF Embryo Gallus domesticus, in aseptic results 24～96 hours, the chick embryo allantoic liquid of the dead and HA>=8log2 of still surviving after 96 hours is as H9 subtype avian influenza virus HZ strain venom;
2) prepare H9 subtype avian influenza virus FJ strain venom: H9 subtype avian influenza virus FJ strain normal saline is done to 10
-4dilution, inoculation 9～11 age in days SPF Embryo Gallus domesticus, in aseptic results 24～96 hours, the chick embryo allantoic liquid of the dead and HA>=8log2 of still surviving after 96 hours is as H9 subtype avian influenza virus FJ strain venom;
3) deactivation H9 subtype avian influenza virus HZ strain venom: adding final concentration in HZ strain venom is 0.2% formalin, 37 ℃ of shaking table deactivations 24 hours;
4) deactivation H9 subtype avian influenza virus FJ strain venom: adding final concentration in FJ strain venom is 0.1% formalin, 37 ℃ of shaking table deactivations 24 hours;
5) prepare oil-phase solution: by white oil and Si Ben-80, according to volume ratio, be 18～20:1 mix homogeneously, after heating for dissolving through 121 ℃ of sterilizings 20 minutes;
6) prepare aqueous phase solution: by the FJ strain venom of the HZ strain venom of deactivation and deactivation, according to volume ratio, be 6:4 mix homogeneously, add the autoclaved tween 80 that accounts for admixture poison liquid volume ratio 0.75%, with magnetic stirring apparatus, mix and be stirred to tween and dissolve completely;
7) emulsifying: be 1～2:1 mix homogeneously by oil-phase solution and aqueous phase solution according to volume ratio, stir in emulsifying mixer, make described bird flu H9 hypotype inactivated vaccine.
As further technical scheme of the present invention, the white oil in step 5) and Si Ben-80 are that 18.5:1 mixes according to volume ratio.
As further technical scheme of the present invention, the oil-phase solution in step 7) and aqueous phase solution are that 1.5:1 mixes according to volume ratio.
The application of a kind of bird flu H9 hypotype inactivated vaccine in prevention avian influenza disease.
Compared to existing technology, beneficial effect of the present invention is: utilize the different local H9 subtype avian influenza strains that separate of 2 strains to be prepared into the inactivated vaccine that a kind of immunogenicity is strong, intersecting protective is good, for preventing chicken H9 subtype avian influenza.After this vaccination chicken, antibody titer is high, makes chicken good to the endemy H9 hypotype of difference strain counteracting toxic substances protection, and safe, effect is stable.
Below in conjunction with the drawings and specific embodiments, the present invention is described in further detail.
Accompanying drawing explanation
Fig. 1 is that the bird flu H9 hypotype antibody level of serum after yellow chicken immune HZ-FJ strain inactivated vaccine changes;
Fig. 2 is that after bamboo silk chicken immune HZ-FJ strain inactivated vaccine, bird flu H9 hypotype antibody level of serum changes.
The specific embodiment
The biological characteristic research of the HZ strain of embodiment 1:H9 subtype avian influenza virus and FJ strain
H9 subtype avian influenza virus HZ strain of the present invention and FJ strain are respectively in the morbidity chicken of China nutrient elements in Huzhou area of Zhejiang Province and Putian, fujian Province area morbidity chicken house is mined massively the pathological material of disease collecting, to be separated to for 2010, through order-checking, identify, and be all bird flu virus H9 hypotype.Its biological characteristic research is as follows: 1) seed culture of viruses goes down to posterity: HZ strain, the primary kind poison of FJ strain (E1 generation) are diluted to 10 with sterile saline
-4in allantoic cavity, inoculate 9 age in days SPF Embryo Gallus domesticus, every embryo 0.2mL, continues to hatch 96 hours in 37 ℃, gather in the crops the Embryo Gallus domesticus of dead in 24～96 hours and survival, after frozen spending the night, gather in the crops one by one Embryo Gallus domesticus liquid and detect its HA and tire, the Embryo Gallus domesticus liquid of aseptic check and tire>=8log2 of HA being mixed to quantitative separating, dated harvest date, Virus passages etc., be labeled as E2 generation, leave and take and continue on a small quantity to go down to posterity, remaining is put-70 ℃ and saves backup.In SPF Embryo Gallus domesticus, be passaged to continuously as stated above the 8th generation (E8 generation), the HA that every generation is measured respectively each embryo tires and average, the results detailed in Table 1.
The HA in table 1 strain E1～E8 generation tires
Result of the test shows, 2 strain AIV H9 hypotype strain HA in the process of going down to posterity all quite stables of tiring, and HA tires more than meansigma methods all remains on 8log2, illustrates that HZ strain and FJ strain all can stably copy and go down to posterity on Embryo Gallus domesticus.
2) virus-specific: by " Chinese veterinary pharmacopoeia " HI test method, HZ strain, FJ strain virus liquid are prepared respectively 4 unit antigens, then carry out HI test with ND, EDS, AIV H5, AIV H7 and AIV H9 standard positive serum, the results detailed in Table 2.
Table 2 strain specific result of the test
Note: ND, EDS, AIV H5, AIV H7, AIV H9 standard positive serum and antigen in the present invention are purchased from China Veterinary Drugs Supervisory Inst. and Harbin veterinary institute.
Result of the test demonstration, the characteristic of HZ strain and FJ strain venom coagulation chicken red blood cell all can be neutralized by H9 subtype avian influenza positive serum specificity, can not be neutralized by ND, EDS and AIV H7 positive serum; There is atomic weak reacting with AIV H5 positive serum, can ignore; Result shows that HZ strain and FJ strain virus liquid all have very strong virus-specific.
3) immunogenicity of virus: the immunogenicity of (1) HZ strain: the E8 of HZ strain is made respectively to oil emulsion inactivated vaccine for seed culture of viruses, 10 of cervical region subcutaneous injection immunity 21 age in days SPF chickens, every 0.3mL, 5 of the identical contrast SPF chickens of the condition of simultaneously establishing.After immunity 21 days, gather chicken serum detect AIV H9 hypotype HI antibody together with matched group, then each intravenous injection HZ strain virus liquid 0.2mL(is containing 2 × 10
6eID
50).After counteracting toxic substances the 5th day, gather every chicken larynx cotton swab, 5 pieces of inoculation 9～10 age in days SPF Embryo Gallus domesticus in allantoic cavity respectively, every embryo 0.2mL, hatches 96 hours for 37 ℃, detects all Embryo Gallus domesticus liquid HA and tires.In the Embryo Gallus domesticus of each cotton swab sample inoculation, as long as there is tire >=4log2 of the HA of 1 piece of Embryo Gallus domesticus, can be judged to virus and separates the positive, virus is separated to negative sample, after a blind passage generation, judge again, the results detailed in Table 3.
The Immunization result of the test of table 3 HZ strain virus
Result of the test demonstration, the HI antibody geometrical mean of immune chicken is 9log2, and contrast chicken HI antibody is 0; After counteracting toxic substances, it is 9/10 that immune group fowl disease poison separates negative rate, and matched group fowl disease poison separation negative rate is 0/5; Show that HZ strain has good immunogenicity.
(2) immunogenicity of FJ strain: detection method is with the detection method of HZ strain, the results detailed in Table 4.
The Immunization result of the test of table 4 FJ strain virus
Result of the test demonstration, the HI antibody geometrical mean of immune chicken is 8.4log2, and contrast chicken HI antibody is 0; After counteracting toxic substances, it is 9/10 that immune group fowl disease poison separates negative rate, and matched group fowl disease poison separation negative rate is 0/5; Show that FJ strain has good immunogenicity.
4) sequence analysis of viral HA gene: with reference to the H9 hypotype AIV sequence data of having delivered, designed and synthesized the RT-PCR primer of AIV HA gene, the HA gene order of HZ strain and FJ strain virus has successfully increased, obtain the HA gene order of HZ strain as shown in SEQ ID NO:1, the HA gene order of FJ strain is as shown in SEQ ID NO:2.
5) antigen correlation analysis: the 6 strain AIV H9 hypotype strains that this laboratory is preserved, comprise HZ strain and FJ strain, be prepared into respectively oil emulsion inactivated vaccine, immunity 21 age in days SPF chickens, in isolated rearing device, prepare single-factor serum, the HI that is used for intersecting test, detects the whether wide spectrum of spectrotype of HZ strain and FJ strain virus.The criterion of antigen dependency is as follows: the dependency between antigen
r is the antigenic specificity between 2 strain virus, r1 be Alphavirus to the HI titre/Alphavirus of second serum the HI titre to first serum, r2 be second virus to HI titre/second virus of first serum the HI titre to second serum; If R=1, shows that between two strains, antigenicity is identical; If 0.67≤R≤1.5, show antigenicity no significant difference between two strains; If 0.5≤R < 0.67, shows that between two strains, antigenicity has little difference; If R < 0.5, shows antigenic obvious difference between two strains; R value is less, and antigenic specificity is larger, the results detailed in Table 5 and table 6.Result of the test demonstration, the antigen dependency of HZ strain and all the other 5 strain AIV H9 viruses is all greater than 0.94, shows that the antigen dependency of this virus and all the other strains is fine; The antigen dependency of FJ strain and all the other 5 strain AIV H9 viruses is all greater than 0.95, shows that the antigen dependency of this virus and all the other strains is fine.
Cross-hemagglutination inhibition test result between table 56 strain virus
Antigen dependency between table 66 strain virus
6) intersection protest test: latter 21 days of HZ strain and the immunity of FJ strain inactivated vaccine; carry out counteracting toxic substances with 6 strain AIV H9 hypotype strains; within after counteracting toxic substances the 5th day, gather chicken larynx cotton swab; in allantoic cavity, inoculation 9～10 age in days SPF Embryo Gallus domesticus carry out virus separation respectively; detect HZ strain and FJ strain and can produce good immune protective effect to the malicious attack in open country, the results detailed in Table 7.
Cross-protection test between table 76 strain strains
The challenge test result of table 7 shows, the intersecting protective of HZ strain is best, and except being 9/11 to autoprotection power and being 8/10 to SN strain protection, it is all protections to all the other 4 strains; FJ strain is also better to the intersecting protective of different strains, viral negative number/counteracting toxic substances number all can reach 8/10 and more than, and can reach respectively 10/11,9/10 to the protection of HZ strain, SN strain.
In conjunction with above test data, the present invention selects HZ strain and FJ strain match and make bird flu H9 hypotype inactivated vaccine.
Embodiment 2: the safety research of bird flu H9 hypotype inactivated vaccine
A preparation method for bird flu H9 hypotype inactivated vaccine, is characterized in that comprising the steps:
1) prepare H9 subtype avian influenza virus HZ strain venom: H9 subtype avian influenza virus HZ strain normal saline is done to 10
-4dilution, inoculation 9～11 age in days SPF Embryo Gallus domesticus, in aseptic results 24～96 hours, the chick embryo allantoic liquid of the dead and HA>=8log2 of still surviving after 96 hours is as H9 subtype avian influenza virus HZ strain venom;
2) prepare H9 subtype avian influenza virus FJ strain venom: H9 subtype avian influenza virus FJ strain normal saline is done to 10
-4dilution, inoculation 9～11 age in days SPF Embryo Gallus domesticus, in aseptic results 24～96 hours, the chick embryo allantoic liquid of the dead and HA>=8log2 of still surviving after 96 hours is as H9 subtype avian influenza virus FJ strain venom;
3) deactivation H9 subtype avian influenza virus HZ strain venom: adding final concentration in HZ strain venom is 0.2% formalin, 37 ℃ of shaking table deactivations 24 hours;
4) deactivation H9 subtype avian influenza virus FJ strain venom: adding final concentration in FJ strain venom is 0.1% formalin, 37 ℃ of shaking table deactivations 24 hours;
5) prepare oil-phase solution: by white oil and Si Ben-80, according to volume ratio, be 18～20:1 mix homogeneously, after heating for dissolving through 121 ℃ of sterilizings 20 minutes;
6) prepare aqueous phase solution: by the FJ strain venom of the HZ strain venom of deactivation and deactivation, according to volume ratio, be 6:4 mix homogeneously, add the autoclaved tween 80 that accounts for admixture poison liquid volume ratio 0.75%, with magnetic stirring apparatus, mix and be stirred to tween and dissolve completely;
7) emulsifying: be 1～2:1 mix homogeneously by oil-phase solution and aqueous phase solution according to volume ratio, with the speed of 10000r/min, stir 10 minutes in emulsifying mixer, make described bird flu H9 hypotype inactivated vaccine.
In order to check the safety of bird flu H9 hypotype inactivated vaccine prepared by the present invention, prepare according to the method described above 2 batches of inactivated vaccines, lot number is respectively 01,02.Inoculate respectively 7 age in days SPF chickens, the yellow chickens of 7 ages in days, 10 every group, this bird flu of the subcutaneous injection H9 of nape portion hypotype inactivated vaccine, 1mL/ is (overdose immunity) only, separately establishes the same terms SPF, yellow chicken does not inoculate in contrast, 5 every group.By observation, inoculate and in latter 14 days, whether occur any part and the general reaction being caused by vaccine and inoculate latter 14 days, 28 vaccine absorbing state, vaccine safety is studied.Wherein, 7 age in days SPF chickens are purchased from the emerging great Hua Nong fowl egg SPF of company limited Experimental Animal Center, and the yellow chicken of 7 ages in days is purchased from Guangdong Wen Shi food Group Plc olive root chicken house.Result of the test is in Table 8 and table 9.
Safety testing result after table 8 SPF chicken immune vaccine
Safety testing result after the yellow chicken immune vaccine of table 9
Above-mentioned result of the test shows, the yellow chicken inoculation of 7 age in days SPF chickens and 7 ages in days bird flu H9 of the present invention hypotype inactivated vaccine, does not observe clinical extremely in 14 days, search for food, drink water and be normal, health condition is good, does not find any part and the systemic adverse reactions that by vaccine, are caused.Inoculate latter 14 days and cut open inspection injection site, the visible a small amount of granule of more than 4/5 inoculation Carnis Gallus domesticus eye is not absorbed completely; Inject and within latter 28 days, cut open inspection, 5/5 inoculation chicken vaccine has absorbed completely, and injection site is showed no the abnormal response being caused by vaccination.Therefore, bird flu H9 hypotype inactivated vaccine of the present invention has good safety.
Embodiment 3: bird flu H9 hypotype inactivated vaccine is to SPF chicken immune potency test
According to the method for embodiment 2, prepare bird flu H9 hypotype inactivated vaccine, by the bird flu H9 hypotype inactivated vaccine immunity 21 age in days SPF chickens of preparation, 0.3mL/ only, exempts from blood sampling in latter 21 days, separation of serum, measure HI antibody titer, carry out under wing vein simultaneously and attack the different local isolated strains of AIV H9 hypotype, counteracting toxic substances dosage be 0.2mL/ only, after counteracting toxic substances the 5th day, gather every chicken larynx swab, through allantoic cavity inoculation 9～10 age in days SPF Embryo Gallus domesticus, carry out virus respectively and separate.5 pieces of throat swab sample allantoic cavity inoculation 9～10 age in days SPF Embryo Gallus domesticus of every chicken, hatch and observe 96 hours, no matter dead germ, the embryo of living all should be measured Embryo Gallus domesticus liquid red cell agglutination valency, there to be 1 piece of Embryo Gallus domesticus liquid red cell agglutination valency to be not less than 4log2(micromethod in 5 pieces of Embryo Gallus domesticus) be judged to infection.To the negative Embryo Gallus domesticus of viral infection, blind passage once.The concrete grouping of test refers to table 10, the results are shown in Table 11.
Table 10 SPF chicken immune challenge test grouping situation
The Immunization protection situation of table 11 vaccine to SPF chicken
Above-mentioned result of the test shows; after SPF chicken immune bird flu H9 hypotype inactivated vaccine the 21st day, produced higher bird flu H9 hypotype serum antibody, in the face of the attack of the different local strains of bird flu H9 hypotype; all produced good immune protective efficiency, 10 chickens are all protected more than 9.
Embodiment 4: after yellow chicken immune bird flu H9 hypotype inactivated vaccine, HI antibody Fluctuation is measured
According to the method for embodiment 2, prepare bird flu H9 hypotype inactivated vaccine, get 32 of the yellow chickens of 21 ages in days, be equally divided into 2 groups, 16 every group.Only, same dosage is pressed at interval after 2 weeks booster immunization is once again for the 1st group of inoculation bird flu H9 hypotype inactivated vaccine 0.3mL/; The 2nd group of not vaccination is as negative control.2 groups of H9 subtype avian influenza antibody horizontals of results of regular determination later, analyze immune group antibody change, and result of the test is shown in Fig. 1, and in figure, HZ-FJ inactivated vaccine represents bird flu H9 hypotype inactivated vaccine.
The result of Fig. 1 shows, after yellow chicken immune bird flu H9 hypotype inactivated vaccine, after exempting from, within 7th, start to produce immunne response, two exempt from after antibody horizontal on the 14th rise rapidly, and after two exempt from, reach antibody peak in 14～28 days, until two exempt from still can to maintain 8 log2 in latter 64 days more than.
Embodiment 5: after bamboo silk chicken immune bird flu H9 hypotype inactivated vaccine, HI antibody Fluctuation is measured
According to the method for embodiment 2, prepare bird flu H9 hypotype inactivated vaccine, get 32 of 14 age in days bamboo silk chickens, be equally divided into 2 groups, 16 every group.Only, same dosage is pressed at interval after 2 weeks booster immunization is once again for the 1st group of inoculation bird flu H9 hypotype inactivated vaccine 0.3mL/; The 2nd group of not vaccination is as negative control.Measure weekly 2 groups of H9 subtype avian influenza antibody horizontals later, analyze immune group antibody change, result of the test is shown in Fig. 2, and in figure, HZ-FJ inactivated vaccine represents bird flu H9 hypotype inactivated vaccine.
The result of Fig. 2 shows, when bamboo silk chicken was exempted from the same day (14 age in days) at head, in body, still there is the bird flu H9 hypotype maternal antibody of certain level, reach 4 log2, along with the growth of chicken age in days, its maternal antibody is reduced to and is tending towards 0 rapidly, immune group chicken is exempted from latter the 7th day since one, the new bird flu H9 serum antibody producing substantially linearly rises, exempts from the 7th day afterwards to two, and vaccine-induced serum antibody reaches peak value, up to 11 log2, then tend towards stability gradually, to two exempt from after the 49th day, more than antibody horizontal still can maintain 8 log2.
Above-mentioned embodiment is only the preferred embodiment of the present invention; can not limit the scope of protection of the invention with this, the variation of any unsubstantiality that those skilled in the art does on basis of the present invention and replacement all belong to the present invention's scope required for protection.
Claims (5)
1. a bird flu H9 hypotype inactivated vaccine, it is characterized in that: by 2 strain H9 subtype avian influenza virus, after deactivation, mix and form with oily adjuvant, 2 strain H9 subtype avian influenza virus are respectively HZ strain and FJ strain, its preservation registration number is respectively CGMCC:8355 and CGMCC:8354, the HA gene order of HZ strain is as shown in SEQ ID NO:1, and the HA gene order of FJ strain is as shown in SEQ ID NO:2.
2. a preparation method for bird flu H9 hypotype inactivated vaccine claimed in claim 1, is characterized in that comprising the steps:
1) prepare H9 subtype avian influenza virus HZ strain venom: H9 subtype avian influenza virus HZ strain normal saline is done to 10
-4dilution, inoculation 9 ~ 11 age in days SPF Embryo Gallus domesticus, in aseptic results 24 ~ 96 hours, the chick embryo allantoic liquid of the dead and HA>=8log2 of still surviving after 96 hours is as H9 subtype avian influenza virus HZ strain venom;
2) prepare H9 subtype avian influenza virus FJ strain venom: H9 subtype avian influenza virus FJ strain normal saline is done to 10
-4dilution, inoculation 9 ~ 11 age in days SPF Embryo Gallus domesticus, in aseptic results 24 ~ 96 hours, the chick embryo allantoic liquid of dead and HA>=8 log2 of still surviving after 96 hours is as H9 subtype avian influenza virus FJ strain venom;
3) deactivation H9 subtype avian influenza virus HZ strain venom: adding final concentration in HZ strain venom is 0.2% formalin, 37 ℃ of shaking table deactivations 24 hours;
4) deactivation H9 subtype avian influenza virus FJ strain venom: adding final concentration in FJ strain venom is 0.1% formalin, 37 ℃ of shaking table deactivations 24 hours;
5) prepare oil-phase solution: by white oil and Si Ben-80, according to volume ratio, be 18 ~ 20:1 mix homogeneously, after heating for dissolving through 121 ℃ of sterilizings 20 minutes;
6) prepare aqueous phase solution: by the FJ strain venom of the HZ strain venom of deactivation and deactivation, according to volume ratio, be 6:4 mix homogeneously, add the autoclaved tween 80 that accounts for admixture poison liquid volume ratio 0.75%, with magnetic stirring apparatus, mix and be stirred to tween and dissolve completely;
7) emulsifying: be 1 ~ 2:1 mix homogeneously by oil-phase solution and aqueous phase solution according to volume ratio, stir in emulsifying mixer, make described bird flu H9 hypotype inactivated vaccine.
3. the preparation method of a kind of bird flu H9 hypotype inactivated vaccine according to claim 2, is characterized in that: the white oil in step 5) and Si Ben-80 are that 18.5:1 mixes according to volume ratio.
4. the preparation method of a kind of bird flu H9 hypotype inactivated vaccine according to claim 2, is characterized in that: the oil-phase solution in step 7) and aqueous phase solution are that 1.5:1 mixes according to volume ratio.
5. a bird flu H9 hypotype inactivated vaccine is in the application preventing in avian influenza disease.
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| CN106085966A (en) * | 2016-06-05 | 2016-11-09 | 青岛易邦生物工程有限公司 | A kind of avian influenza strain |
| CN106075424A (en) * | 2016-06-05 | 2016-11-09 | 青岛易邦生物工程有限公司 | One avian influenza virus 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 |
| 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 |
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| CN106085966A (en) * | 2016-06-05 | 2016-11-09 | 青岛易邦生物工程有限公司 | A kind of avian influenza strain |
| CN106075424A (en) * | 2016-06-05 | 2016-11-09 | 青岛易邦生物工程有限公司 | One avian influenza virus vaccine |
| CN106075424B (en) * | 2016-06-05 | 2019-10-25 | 青岛易邦生物工程有限公司 | One avian influenza virus 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 |
| 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 |
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Inventor after: Chen Ruiai Inventor after: Lin Qiping Inventor after: Chen Chunli Inventor after: Zhang Wenyan Inventor after: Huang Wenke Inventor after: Xu Jiahua Inventor after: Liu Jinrong Inventor after: Yan Jiezhen Inventor after: Gao Yan Inventor before: Chen Ruiai Inventor before: Lin Qiping Inventor before: Chen Chunli Inventor before: Huang Wenke Inventor before: Xu Jiahua Inventor before: Yan Jiezhen |