CN101450207A - Human influenza-poultry influenza combined vaccine and preparation method thereof - Google Patents

Abstract

The invention provides a quadrivalence combined vaccine comprising H1N1, H3N2, B types flu and H5N1 type avian influenza and a preparing method. Concentration of each vaccine hemagglutinin is calculated with hemagglutinin: H1N1 type flu vaccine 20-40 mcg/mL, H3N2 type flu vaccine 20-40 mcg/mL, B type flu vaccine 20-40 mcg/mL and H5N1 type flu avian influenza 20-40 mcg/mL. The vaccine combining with human flu and avian influenza for human can solves problem of difficult to vaccination avian influenza vaccine, prevents avian influenza at the same time prevents huamn flu. Four indications (egg albumen, endotoxin, total protein contents, rate of total protein and hemagglutinin) The combined vaccine closly-related with side effect in the combined vaccine has low flu preparing regulation than national regulation, and has higher safety.

Description

A kind of human influenza-poultry influenza combined vaccine and preparation method thereof

(1) technical field

The present invention relates to a kind of human influenza-poultry influenza combined vaccine and preparation method thereof, especially a kind of combined vaccine that comprises H1N1 type, H3N2 type, Type B influenza and H5N1 type avian influenza vaccine and preparation method thereof.

(2) background technology

On October 14th, 2005, WHO appealed to the world: " whole world is in the edge of another time flu outbreak at present; in case take place; propagate rapidly; will be infected less than 3 months All Countries; underestimate death toll 2,000,000 to 7,400,000; economic and society destroys huge, and each country of the whole world all must do some preparations ".

On JIUYUE 10th, 2003 1, and H5N1 bird flu case 387 people are taken place all over the world altogether, dead 245 people, case fatality rate is up to 63.3%, potential hazard surpasses SARS, the health that is threatening the people of the world, and the development of human-used avian influenza vaccine has caused the great attention of national governments.

According to statistics international pharmacy corporation community of association (IFPMA) in January, 2006, comprise that 12 (14) units of states such as method, American and Britain, moral, day, Australia, Russia, Belgium, (China) have carried out the research of human H5N1 vaccine to global avian influenza vaccine anthroposcopy.In April, 07, the U.S. is the H5N1 vaccine listing of first approval Pasteur S.A. in the world, and the said firm announces, will donate 6,000 ten thousand doses of H5N1 vaccines to World Health Organization (WHO) in 3 years and be used for global reserves.China also ratifies Ke Xing company in June, 08 and produces avian influenza vaccine, as emergent during the course of the Olympic Games stock vaccine.

The difficult problem that present avian influenza vaccine manufacturer runs into is the question of market.It is generally acknowledged: bird flu patient surrounding population and close contact birds person should inoculate this vaccine, and need to carry out prophylactic immunization for broad masses of the people? this question answering is negated, because can H5N1 virus cause human-to-human transmission, be a unknown number, generally inoculation, cost is too high, thereby at present only as technical reserve, or goods and materials stock, because product does not have market, have influenced the enthusiasm that manufacturer invests and develops avian influenza vaccine.

Now, the crowd inoculates the seasonal current influenza vaccine and constantly enlarges, and especially developed country is comparatively general, reaches 27% as U.S.'s rate of vaccination, and European countries' rate of vaccination does not wait 7.8%～17.7%.China also constantly enlarges, if add human-used avian influenza vaccine in the human influenza vaccine, cost is not high easily to be accepted by people again.

(3) summary of the invention

The object of the invention provides a kind of human influenza-poultry influenza combined vaccine, adds human-used avian influenza vaccine in the human influenza vaccine, and cost is not high easily to be accepted by people again, and has solved the problem of avian influenza vaccine inoculation difficulty.

The technical solution used in the present invention is:

A kind of human influenza-poultry influenza combined vaccine, mainly comprise H1N1 type human influenza vaccine, H3N2 type human influenza vaccine, Type B human influenza vaccine and H5N1 type human and bird fluenza vaccine, each vaccine in hemagglutinin concentration is: H1N1 type human influenza vaccine 20～40 μ g/mL, H3N2 type human influenza vaccine 20～40 μ g/mL, Type B human influenza vaccine 20～40 μ g/mL and H5N1 type human and bird fluenza vaccine 20～40 μ g/mL.More than be the main effective ingredient of combined vaccine, its auxiliary element can be selected according to this area conventional method as solvent buffer, stabilizing agent, antiseptic etc.

The purpose of research people-bird flu combined vaccine is the inoculation by this vaccine, make the crowd obtain the fundamental immunity of anti-avian influenza virus, in case infect, may not fall ill or the state of an illness alleviates, as present H1N1 influenza, though it is still popular in the crowd, but it is fearful doomed dead ten million people of seasonal disease to be very popular unlike " spanish influenza " that H1N1 virus in 1918 causes, so human influenza-human-used avian influenza combined vaccine, can solve the problem of avian influenza vaccine inoculation difficulty, in the prevention human influenza, the prevention that has also solved bird flu.Therefore the present invention has changed present situation to the passive prevention of bird flu, the strategy that transfers active prevention to, because occupying soon first of the virosis of influenza spread speed, in case the H5N1 bird flu virus obtains the ability of human-to-human transmission, in 1～February, can spread all over the whole world, so often effect is undesirable in passive prevention, because will produce the avian influenza vaccine that can satisfy global demand at short notice, hardly may.

Also can contain antiseptic phenoxyethanol 4～6mg/mL in the described combined vaccine.Thimerosal is used as antiseptic widely in China's traditional vaccine.In the thimerosal 49.6% is ethyl hydrargyrum, and it has very high neurotoxicity.Before 1986, it is 100 micrograms that 2 years old child accepts the total amount of ethyl hydrargyrum.By 1991, owing to contain the hepatitis B of thimerosal and the inoculation of influenza vaccines, the total amount of hydrargyrum increased to 246 micrograms, was original 2.5 times.Hydrargyrum is mainly discharged from urine, because child's kidney incomplete development still, the age, float hydrargyrum ability was poor more more, if the child constantly the mercurous influenza of inoculation, encephalitis b and and vaccine such as epidemic encephalitis, the savings that may cause hydrargyrum is poisoned, and thinks that now child's autistic morbidity is relevant with mercurialism.Thereby the vaccine that contains thimerosal is forbidden by the Iowa of the U.S., California and Britain and other European countries.The present invention selects for use phenoxyethanol as antiseptic, and its toxicity is low, belongs to non-dangerous article, and is safe.To recommend to contain in the described combined vaccine volume final concentration be that 0.004%～0.007% tween 80 is as stabilizing agent in the present invention in addition.

Each vaccine in hemagglutinin concentration is: H1N1 type human influenza vaccine 30 μ g/mL, H3N2 type human influenza vaccine 30 μ g/mL, Type B human influenza vaccine 30 μ g/mL and H5N1 type human and bird fluenza vaccine 30 μ g/mL.

Described human influenza-poultry influenza combined vaccine is the diluent preparation with the phosphate buffer of 0.01mol/L, pH7.2.

The human influenza H1N1/IVR-145 seed culture of viruses that described H1N1 type human influenza vaccine is recommended or approved from WHO.The human influenza H3N2/NYMC X-161B seed culture of viruses that described H3N2 type human influenza vaccine is recommended or approved from WHO.The human influenza B/Malysia-2506-2004 seed culture of viruses that described Type B human influenza vaccine is recommended or approved from WHO.The H 5 N 1 avian influenza attenuated strain seed culture of viruses that described H5N1 human and bird fluenza vaccine is recommended or approved from WHO.

Described combined vaccine can obtain easily by this area conventional method, main route is as follows: virus is planted Embryo Gallus domesticus → cultivation results → centrifugal precipitation → ultrafiltration and concentration → third lactone deactivation → band centrifugation → ultrafiltration desaccharide → column chromatography → cracking → band centrifugation → ultrafiltration desaccharide → filtration → unit price stock solution of going, dilution makes the mixing of unit price virus stock solution used according to the method described above respectively after can obtain combined vaccine.Described combined vaccine can not add adjuvant, also can add aluminium hydroxide as adjuvant.

Combined vaccine preparation method of the present invention is preferably carried out as follows:

(1) human influenza H1N1/IVR-145 seed culture of viruses, human influenza H3N2/NYMC X-161B seed culture of viruses, human influenza B/Malysia-2506-2004 seed culture of viruses and H 5 N 1 avian influenza attenuated strain seed culture of viruses are produced univalent vaccine stock solution by following step respectively:

1. seed culture of viruses is inoculated in the chick embryo allantois intracavity after going down to posterity, diluting, and cultivates 48～72 hours for 33～35 ℃;

2. screen the live chickens embryo and put 2～8 ℃ of cold embryos 12～20 hours, draw the clarifying allantoic fluid of outward appearance and add formalin, put 2～8 ℃ and descended 18～20 hours, collect viral liquid, 40～50 times of ultrafiltration and concentration; Need sampling to carry out sterility test and hemagglutinative titer mensuration, require results liquid sterility test qualified, hemagglutinative titer is not less than 1:320.

3. the viral liquid after concentrating adds β-third lactone of viral liquid long-pending 1/4000,2～8 ℃ of following deactivations 18～20 hours;

4. the viral liquid after the deactivation purified after, add final concentration and be 0.4% Triton X-100 or/and final concentration 0.4% NaTDC as decomposition agent, mix the back and handle 2～4min with the ultrasonic cell disruptor of 600W～700W power, put 20～30 ℃ again and act on 90～120 minutes down, obtain the virolysis thing;

5. the virolysis thing purified after, the Tween 80 that adds volume and be lysate volume 0.006% behind the purification is as stabilizing agent, membrane filtration obtains univalent vaccine stock solution;

(2) will make univalent vaccine stock solution is mixed in proportion, phosphate buffer with 0.01mol/L, pH7.2 is diluted to H1N1 type human influenza vaccine concentration 20～40 μ g/mL, H3N2 type human influenza vaccine concentration 20～40 μ g/mL, Type B human influenza vaccine concentration 20～40 μ g/mL and H5N1 type human and bird fluenza vaccine concentration 20～40 μ g/mL by hemagglutinin content, and to add dense eventually be that the phenoxyethanol of 4～6mg/mL is as antiseptic, membrane filtration obtains described human influenza-poultry influenza combined vaccine.

Concrete, described combined vaccine can obtain as follows:

(1) virus inoculation and cultivation: get work seed lot seed culture of viruses, melt the PBS dilution 10 of back with 0.01mol/L ³-10 ⁵Doubly, and add the kanamycin of 500 μ g/ml.In the chick embryo allantois intracavity, every embryo 0.2ml inoculates rearmounted 33～35 ℃ of cultivations, 48～72 hours with the virus inoculation after the dilution.Work seed lot seed culture of viruses melts the back if once do not use up, and must not freeze to use again.Must not add penicillin or other beta-lactam antibiotics in the production of vaccine process.

(2) virus results: screening live chickens embryo was put 2～8 ℃ of cold embryos 12～20 hours, drew the clarifying allantoic fluid of outward appearance in sterile chamber, added final concentration and was 0.04% ratio and add formalin, put 2～8 ℃ 8～20 hours down.Sterility test is carried out in sampling and hemagglutinative titer is measured, and requires results liquid sterility test qualified, and hemagglutinative titer is not less than 1:320.

(3) virus results liquid concentrates: the results liquid after the deactivation is with the rotating speed of 13000～15000rpm/min, carry out the continuous flow centrifugation clarification with the flow that is no more than 1500ml/min, be 40～50 times of the ultrafilter membrane ultrafiltration and concentration of 1,000,000 molecular weight with the aperture then, inlet pressure is no more than 0.2Mpa during ultrafiltration.

(4) inactivation of virus: add β-third lactone of 1/4000 (v/v) in the viral liquid after concentrating, put 2～8 ℃ of deactivations 18～20 hours.

(5) viral purification: sucrose density band centrifugation: the viral liquid after concentrating is with sample on the amount that is no more than centrifugal chamber volume 50%, in 0～55% sucrose solution with the rotating speed gradient centrifugation of 30000rpm/min 3 hours, under the ultraviolet monitoring of 280nm wavelength, collect the 3rd peak (sucrose concentration is 30%～42% liquid section), to extract virus, on ultrafilter, carry out ultrafiltration then and clean, to remove sucrose with PBS.

The ultrafiltration cleaning method is: the viral liquid that will extract dilutes 2～4 times with the PBS of 0.01mol/L (pH7.2), the ultrafilter membrane of reuse 1,000,000 molecular weight carries out ultrafiltration and concentration, and PBS is added on ultrafiltration limit, limit, make the liquid measure during the ultrafiltration keep constant, and the total liquid measure of PBS that ultrafiltration is cleaned is no less than 4 times of the viral liquid of carrying.

(6) column chromatography: carry out chromatography purification with the Sephrose-4FF gel, the applied sample amount of chromatography is no more than 8% of gel volume, eluent is the PBS (pH7.2) of 0.01mol/L, and elution flow rate is 5-6mm/min, collects first peak under the ultraviolet monitoring of 280nm wavelength.

(7) virolysis: the viral liquid behind the purification adds final concentration be 0.4%Triton X-100 or/and 0.4% NaTDC as decomposition agent, 2～4min is handled with the ultrasonic cell disruptor of 600W～700W power in the mixing back, puts 20～30 ℃ again and acts on 90～120 minutes down.

(8) purification behind the virolysis: the virolysis thing is with sample on the amount that is no more than centrifugal chamber volume 50%, in 0～55% sucrose solution with the rotating speed gradient centrifugation of 30000r/min 2 hours, under the ultraviolet monitoring of 280nm wavelength, remove first peak and last peak then, the collection sucrose concentration is 2%～42% liquid section, on ultrafilter, carry out ultrafiltration again and clean, to remove sucrose with PBS.

The ultrafiltration cleaning method is: use the PBS of 0.01mol/L (pH7.2) to dilute 2～4 times the lysate after centrifugal, the ultrafilter membrane of reuse 100,000 molecular weight carries out ultrafiltration and concentration, and add PBS while concentrating, make the liquid measure during the ultrafiltration keep constant, and the total liquid measure of PBS that ultrafiltration is cleaned is no less than 6 times of lysate.

Lysate behind the purification adds the tween 80 of 0.006% (v/v), and the membrane filtration through 0.22 μ m aperture is unit price stock solution, should place 2～8 ℃ of preservations.

(9) combined vaccine preparation: it is respectively 20～40 μ g/ml that various unit price stock solution is diluted to by hemagglutinin content, diluent is the PBS (pH7.2) of 0.01mol/L, and add dense eventually be the phenoxyethanol of 4～6mg/ml as antiseptic, the membrane filtration in 0.22 μ m aperture is described combined vaccine.

The 3 valency combined vaccines that the human influenza vaccine is made up of influenza A virus A1 (H1N1), A3 (H3N2) and Type B virus originally, and H5N1 and they belong to together of the same race, belong to the A type with A1 (H1N1), A3 (H3N2), nearer than Type B, thereby in the human influenza vaccine, add the H5N1 composition, do not have bad influence in theory each other.According to grasp to the influenza virus purification technique, can accomplish in the combined vaccine with closely-related 4 indexs of the side reaction ratio of hemagglutinin (content of egg protein, endotoxin, total protein, total protein with) lowly more than the influenza producting rule of country, this is the material base that guarantees the combined vaccine safety.

Beneficial effect of the present invention is mainly reflected in: 4 valency combined vaccines of a kind of H1N1 of comprising type, H3N2 type, Type B influenza and H5N1 type avian influenza vaccine and preparation method thereof are provided, with inventor's influenza-human-used avian influenza combined vaccine, can solve the problem of avian influenza vaccine inoculation difficulty, in the prevention human influenza, the prevention that has also solved bird flu; According to the inventive method, make close in the vaccine with closely-related 4 indexs of the side reaction ratio of hemagglutinin (content of egg protein, endotoxin, total protein, total protein with) low, safe more than the influenza producting rule of country.

(4) description of drawings

Fig. 1 is the R1194 strain hemagglutinin nucleotide sequence that records;

Fig. 2 is R1194 strain hemagglutinin nucleotide sequence and original strain nucleotide sequence difference;

Fig. 3 is that R1194 strain hemagglutinin nucleotide sequence and original strain aminoacid sequence change;

Fig. 4 be before the R1194 virolysis electromicroscopic photograph (* 60K);

Fig. 5 be behind the R1194 virolysis electromicroscopic photograph (* 60K);

Fig. 6 is the SDS-PAGE electrophoresis result before and after the R1194 virolysis; M:mark; 1: before the cracking; 2: after the cracking-A; 3～7: after the cracking-B;

Fig. 7 is the figure as a result with the primer amplification vaccine of making a definite diagnosis the H5N1 bird flu virus; M:Mark; 1: vaccine.

(5) specific embodiment

The present invention is described further below in conjunction with specific embodiment, but protection scope of the present invention is not limited in this:

Embodiment 1:

The acquisition of combined vaccine:

Use Embryo Gallus domesticus 1.1 produce

Seed culture of viruses goes down to posterity and prepares with Embryo Gallus domesticus and should derive from SPF chicken group; Production of vaccine should derive from the healthy chicken flock of raising in the closed room with Embryo Gallus domesticus.

1.2 seed culture of viruses

1.2.1 title and source

The bird flu seed culture of viruses: the NIBRG-14 strain derives from Britain national biological product calibrating institute (NIBSC); Form by the method transformation of A/VietNam/1194/2004 H5N1 strain, be called for short the R1194 strain through anti-phase heredity.

Influenza seed culture of viruses: WHO recommends to be used for strain---A1/IVR-145, A3/NYMC X-161B, the B/Malysia-2506-2004 that the 2007-2008 influenza vaccines produce.

1.2.2 the foundation of seed lot

Should meet the current edition " requirement of three general rules of Chinese pharmacopoeia " biological product production is examined and determine with bacterium kind rule of management ".

1.2.3 the calibrating of seed lot seed culture of viruses

Main seed lot should carry out following comprehensive calibrating, and the work seed lot should carry out 1.2.3.1～1.2.3.5 item calibrating at least.

1.2.3.1 discrimination test

Carry out single immunodiffusion test with corresponding (Asia) type specificity immune serum by 3.1.4, should produce the specificity precipitation, prove that the antigenicity of sample is consistent with the recommendation Strain.

1.2.3.2 hemagglutinative titer

Adopt direct blood clotting method to detect, hemagglutinative titer should be not less than 1:320.。

Sample thief 25uL carries out doubling dilution with normal saline in 96 hole blood-coagulation-boards, each dilution factor adds equivalent 1% chicken erythrocyte suspension, fully mixing is placed in 4～8 ℃ and was judged in 30～60 minutes, and the chicken red blood cell with 50% is judged to hemagglutinative titer by agglutinative high dilution.

1.2.3.3 titration of virus

Measure virus titer with Embryo Gallus domesticus infection method, should be not less than 7.0Log EID ₅₀/ mL.

The seed culture of viruses sample is made 10 times of serial dilutions with broth medium, and (the lyophilizing seed culture of viruses dissolves earlier and is diluted to 1ml, and its dilution factor is 10 ^-1), select 10 ^-4～10 ^-9Dilution factor is respectively inoculated 4 9～11 age in days chick embryo allantoic cavities, and every embryo inoculum concentration 0.2ml puts 34～35 ℃ and cultivated 72 hours, and Embryo Gallus domesticus dead in 24 hours should be discarded, and every embryo is taken a sample respectively and done direct hemagglutination test, calculates virus titer EID according to the male Embryo Gallus domesticus number of blood clotting ₅₀, the negative logarithm that infects high dilution with the half Embryo Gallus domesticus is a virus titer.

1.2.3.4 sterility test

Check in accordance with the law, should be up to specification.

1.2.3.5 mycoplasma inspection

Check in accordance with the law, should be up to specification.

1.2.3.6 exogenous avian leukosis virus detects

The antiserum mixed in equal amounts of influenza seed culture of viruses and corresponding (Asia) type, the fibroblast of inoculation 9 age in days SPF Embryo Gallus domesticus is put 5%CO in 37 ℃ and after 1 hour ₂In 4 generations of 37 ℃ of continuous culture of incubator, in 7 days per generations, establish the positive and negative control simultaneously.Adopt the ELISA method to detect culture, testing result should be negative.

1.2.3.7 exogenous aviadenovirus detects

The antiserum mixed in equal amounts of influenza seed culture of viruses and corresponding (Asia) type in 37 ℃ and be inoculated in the cell monolayer for preparing with 13 age in days SPF Embryo Gallus domesticus livers after 1 hour, is put 5%CO ₂In 4 generations of 37 ℃ of continuous culture of incubator, in 7 days per generations, establish the positive and negative control simultaneously.With I type and the exogenous aviadenovirus of III type in the serological method inspection culture, testing result should be negative respectively.

1.2.4 seed culture of viruses goes down to posterity

Get seed culture of viruses and redissolve, be diluted to 10 to 1mL ³～10 ⁵ Back inoculation 9～11 age in days SPF chick embryo allantoic cavities, inoculum concentration 0.2mL/ embryo, cultivate after 72 hours the results allantoic fluid and mix for 33～35 ℃, add behind the equivalent skimmed milk and be stored in below-70 ℃ after packing (0.2mL/ props up) lyophilizing, be main through assay approval for seed lot.The seed culture of viruses that takes out from main seed lot goes down to posterity as stated above, and results liquid packing is also frozen in being the work seed lot below-20 ℃.The strain self-separation rises to the vaccine generation of producing must not surpass for 15 generations, and manufacturing enterprise must not go down to posterity to the work seed lot of making and surpassed for 4 generations Zi obtaining seed culture of viruses.

1.2.5 seed culture of viruses is preserved

The lyophilizing seed culture of viruses is being preserved below-70 ℃; The liquid seed culture of viruses is being preserved below-20 ℃.

1.3 the acquisition of unit price stock solution:

1.3.1 Embryo Gallus domesticus is prepared

Select clear, the activated Embryo Gallus domesticus of no deformity, blood vessel of 9～11 ages in days for use.

1.3.2 virus inoculation and cultivation

Get work seed lot seed culture of viruses, melt the PBS dilution 10 of back with 0.01mol/L ⁵Doubly, and add the kanamycin of 500 μ g/mL.In the chick embryo allantois intracavity, every embryo 0.2mL inoculates rearmounted 33～35 ℃ and cultivated 72 hours with the virus inoculation after the dilution.Work seed lot seed culture of viruses melts the back if once do not use up, and must not freeze to use again.Must not add penicillin or other beta-lactam antibiotics in the production of vaccine process.

1.3.3 virus results

Screening live chickens embryo was put 2～4 ℃ of cold embryos 18 hours, drew the clarifying allantoic fluid of outward appearance in sterile chamber, added final concentration and was 0.04% ratio and add formalin, put 2～8 ℃ following 20 hours.Sterility test is carried out in sampling and hemagglutinative titer is measured, and requires results liquid sterility test qualified, and hemagglutinative titer is not less than 1:320.

1.3.4 virus results liquid concentrates

Results liquid after the deactivation carries out the continuous flow centrifugation clarification with the rotating speed of 15000rpm/min and the flow that is no more than 1500mL/min, is 50 times of the ultrafilter membrane ultrafiltration and concentration of 1,000,000 molecular weight with the aperture then, and inlet pressure is no more than 0.2Mpa during ultrafiltration.

1.3.5 inactivation of virus

Add β-third lactone of 1/4000 (v/v) in the viral liquid after concentrating, put 2～8 ℃ of deactivations 20 hours.

1.3.6 viral purification

1.3.6.1 sucrose density band centrifugation

Viral liquid after concentrating is with sample on the amount that is no more than centrifugal chamber volume 50%, in 0～55% sucrose solution with the rotating speed gradient centrifugation of 30000rpm/min 3 hours, under the ultraviolet monitoring of 280nm wavelength, collect the 3rd peak (sucrose concentration is 30%～42% liquid section), to extract virus, on ultrafilter, carry out ultrafiltration then and clean, to remove sucrose with PBS.

The ultrafiltration cleaning method is: the viral liquid that will extract dilutes 4 times with the PBS of 0.01mol/L (pH7.2), the ultrafilter membrane of reuse 1,000,000 molecular weight carries out ultrafiltration and concentration, and PBS is added on ultrafiltration limit, limit, make the liquid measure during the ultrafiltration keep constant, and the total liquid measure of PBS that ultrafiltration is cleaned is no less than 4 times of the viral liquid of carrying.

1.3.7.2 column chromatography

Carry out chromatography purification with the Sephrose-4FF gel, the applied sample amount of chromatography is no more than 8% of gel volume, and eluent is the PBS (pH7.2) of 0.01mol/L, and elution flow rate is 5～6mm/min, collects first peak under the ultraviolet monitoring of 280nm wavelength.

1.3.8 virolysis

Viral liquid behind the purification add final concentration be 0.4%Triton X-100 and 0.4% NaTDC as decomposition agent, mix the back and handle 4min with the ultrasonic cell disruptor of 600W～700W power, put 20～30 ℃ of effects 120 minutes down again.

1.3.9 the purification behind the virolysis

The virolysis thing is with sample on the amount that is no more than centrifugal chamber volume 50%, in 0～55% sucrose solution with the rotating speed gradient centrifugation of 30000r/min 2 hours, under the ultraviolet monitoring of 280nm wavelength, remove first peak and last peak then, the collection sucrose concentration is 2%～42% liquid section, on ultrafilter, carry out ultrafiltration again and clean, to remove sucrose with PBS.

The ultrafiltration cleaning method is: use the PBS of 0.01mol/L (pH7.2) to dilute 2～4 times the lysate after centrifugal, the ultrafilter membrane of reuse 100,000 molecular weight carries out ultrafiltration and concentration, and add PBS while concentrating, make the liquid measure during the ultrafiltration keep constant, and the total liquid measure of PBS that ultrafiltration is cleaned is no less than 6 times of lysate.

1.3.10 filter

Lysate behind the purification adds the Tween 80 of 0.006% (v/v), and the membrane filtration through 0.22 μ m aperture is unit price stock solution.

1.3.11 stock solution is preserved

Should place 2～8 ℃ of preservations.

1.4 combined vaccine semi-finished product

1.4.1 combined vaccine preparation

Various unit price stock solution is diluted to 30 μ g/mL by hemagglutinin content, and the dilution liquor is the PBS (pH7.2) of 0.01mol/L, and add dense eventually be the phenoxyethanol of 5mg/mL as antiseptic, the membrane filtration in 0.22 μ m aperture is the combined vaccine semi-finished product.

1.5 combined vaccine finished product

1.5.1 in batches

Should meet the current edition " requirement of three general rules of Chinese pharmacopoeia " biological product are rules in batches ".

1.5.2 packing

Should meet the current edition " regulation of three general rules of Chinese pharmacopoeia " biological product packing and lyophilizing rules ".

1.5.3 specification

Every bottle of (propping up) 0.5mL.Per 1 human dosage is 0.5mL, contains each influenza virus strain hemagglutinin and should be not less than 15 μ g.

1.5.4 packing

Should meet current edition " three general rules of Chinese pharmacopoeia " biological product packing rules " regulation.

2, calibrating

2.1 unit price stock solution calibrating

2.1.1 sterility test

Check in accordance with the law, should be up to specification.

2.1.2 inactivation of virus demonstration test

Sample after the deactivation is inoculated in 9～11 age in days chick embryo allantoic cavities by former times and 1:10 and 1:100 dilution back grouping, every embryonic breeding kind 0.2mL, 10 Embryo Gallus domesticus of each dilution factor inoculation are put 33～35 ℃ and were cultivated 72 hours.Dead not counting in 24 hours, every group of Embryo Gallus domesticus survives 80% at least.Every embryo is got the 0.5mL allantoic fluid in the survival Embryo Gallus domesticus, after the group mixing, a blind passage generation again, every group is inoculated in 9～11 age in days chick embryo allantoic cavities, every embryo 0.2mL, 10 Embryo Gallus domesticus of each dilution factor inoculation are put 33～35 ℃ of trainings 72 hours, get allantoic fluid and carry out direct hemagglutination test, hemagglutination should not appear in the result.

2.1.3 discrimination test

Carry out single immunodiffusion test with corresponding (Asia) type specificity immune serum by 3.1.4, should produce the specificity precipitation, prove that the antigenicity of sample is consistent with the recommendation Strain.

2.1.4 hemagglutinin content

Adopt simple immunodiffusion method.

Preparation contains 1% agarose gel plate of viral standard serum, (aperture is 2.5～3mm) in punching, standard antigen and sample to be checked are added with the amount of every hole 10 μ L respectively, put 20～25 ℃ of diffusions at least 21 hours, soak 1 hour after drying, dyeing, decolouring with PBS.Accurate measurement standard antigen and the formed deposit ring diameter of sample to be checked, the diameter and the corresponding antigens content of the precipitation ring that forms with standard antigen carry out rectilinear regression, obtain linear regression equation, the deposit ring diameter of product is examined in substitution, can obtain the content of the hemagglutinin of stock solution.

2.2 combined vaccine semi-finished product calibrating

2.2.1 sterility test

Check in accordance with the law, should be up to specification.

2.2.2 hemagglutinin content

Undertaken by the 2.1.4 item, requirement is 24～36 μ g/ strain/mL.

2.2.3 total protein contains

Measure by " Lowry method ", should not be higher than (420 μ g/mL), and be no more than 4.2 times of the vaccine hemagglutinin content.

2.2.4 egg white protein content

Adopt counter immunoelectrophoresis to detect, should not be higher than 250ng/mL.

The antiovalbumin immune serum added cathode aperture, testing sample add anode hole on 1% agarose gel plate on 1% agarose gel plate, every hole 10uL (aperture 2.5mm, the about 1cm of pitch of holes), on electrophresis apparatus with 120V～150V voltage electrophoresis 35～40min, in water, soaked 1 hour then, carry out drying, dyeing and decolouring again.Do the standard series of 0～1000ng/mL simultaneously, sample determination hole and standard series are contrasted, egg white protein content in observation and the judgement sample.

2.2.5 the Triton X100 determination of residual amount

Residual quantity should not be higher than 100 μ g/mL.

2.2.5 Tween 80 Determination on content

Content should not be higher than 60 μ g/mL.

2.2.6 phenoxyethanol Determination on content

Content should be 4～6m g/mL.

2.2.7 NaTDC Determination on content

Content should not be higher than 100 μ g/mL.

2.3 finished product calibrating

2.3.1 visual examination

The vaccine outward appearance is colourless or the microemulsion white liquid, no foreign body.

2.3.2 loading amount

Check in accordance with the law, should be not less than labelled amount.

2.3.3 sterility test

Check in accordance with the law, should be up to specification.

2.3.4 discrimination test

Undertaken by the 2.1.3 item, the result proves that its antigenicity is consistent with the Strain of recommendation.

2.3.5 hemagglutinin content

Undertaken by the 2.1.4 item, hemagglutinin (HA) content should be (30 ± 6 μ g/mL).

2.3.6?pH

Check that in accordance with the law pH should be 7.0～7.6.

2.3.7 free formaldehyde content

Check in accordance with the law, should not be higher than 20 μ g/mL.

2.3.8 phenoxyethanol content

Content should be 4～6mg/mL.

2.3.9 total protein content

Measure with the Lowry method, should be no more than 420 μ g/mL, and be no more than 4.2 times of the vaccine hemagglutinin content.

2.3.10 egg white protein content

Undertaken by the 2.2.4 item, egg white protein content should not be higher than 250ng/mL.

2.3.11 bacteria endotoxin content is measured

Check in accordance with the law, should not be higher than 50EU/mL.

2.3.12 the undue toxicity checks

Check in accordance with the law, should be up to specification.

Embodiment 2:

One, experiment material and step:

1, vaccine immunity animal experiment

The combined vaccine that makes by embodiment 1 method, warp is to multiple animal immune evidence, chicken and mice sensitivity are very poor, rat is too responsive again, and rabbit is more satisfactory, show that antibody response is relatively stable, amount-validity response is obvious, the moderate human body that approaches of intensity, characteristics such as the big and blood sampling of blood volume is easy.

The rabbit of test usefulness is New Zealand's kind, and about 2 kilograms, male and female are regardless of, from the academy of agricultural sciences, Zhejiang Province.

2, vaccine immunity rabbit test

1) dosage and grouping (each the univalent vaccine stock solution by embodiment 1 method makes with PBS (pH7.2) dilution of 0.01mol/L, is mixed with the unit price or the polyvalent vaccine of following composition, and containing Adjuvanted vaccines interpolation aluminium hydroxide is adjuvant, and its consumption is 0.5mg/mL):

1. each 15 μ g HA/0.5mL does not have adjuvant human influenza 3 valency vaccines.

2. each 15 μ g HA/0.5mL adjuvant human influenzas, 3 valency vaccine.

3. 15 μ g HA/0.5mL do not have adjuvant bird flu univalent vaccine.

4. 15 μ g HA/0.5mL adjuvant bird flu univalent vaccines.

5. each 15 μ g HA/0.5mL does not have adjuvant human influenza-poultry influenza 4 valency Seedlings.

6. each 15 μ g HA/0.5mL adjuvant human influenza-poultry influenzas, 4 valency vaccine.

7. each 15 μ g HA/0.5mL does not have adjuvant human influenza-poultry influenza 4 valency Seedlings.

8. each 15 μ g HA/0.5mL adjuvant human influenza-poultry influenzas, 4 valency vaccine.

9. blank (not inoculating any vaccine).

2) immunization method: every rabbit leg muscle inoculated respectively and organized vaccine 0.5mL, and 5 every group, strengthen a pin with quadrat method after 15 days, totally 2 pins, (7) (8) 2 groups are not strengthened.

3) blood sampling:

In the time of 0,15,30 day, take a blood sample totally 3 times for 1～6 group:

1. the same day (preceding antibody horizontal is exempted from observation) before the first pin;

2. same day before the booster shot (observe behind 1 pin 15 days antibody horizontal);

3. 15 days (observe 2 pins after antibody horizontal) after the booster shot;

In the time of 0,30 day, take a blood sample totally 2 times for 7～8 groups:

1. the same day (preceding antibody horizontal is exempted from observation) before the first pin;

2. behind the first pin 30 days (observe behind 1 pin 30 days antibody horizontal).

Each venous blood collection 5～10mL, separation of serum, frozen below-30 ℃, be used to survey blood and press down antibody and neutralizing antibody.

2, acute toxicity test:

Above-mentioned 6 kinds of vaccines by the biological product rules requirement of China, carry out the acute toxicity safety testing respectively in mice and Cavia porcellus.

4, neutralizing antibody detects:

In the egg embryo, measure fixed virus (10 ³EID ₅₀/ mL), and dilute serum, virus and serum mixed in equal amounts, in 37 ℃ and 90 minutes, every egg embryonic breeding kind 0.2mL (contains 100EID ₅₀), 4 egg embryos of every dilution factor inoculation, the high dilution that 4 complete suppresses is tired for neutralization.

5, hemagglutination inhibition test:

According to a conventional method, hemagglutinin is with the self-control of R1194 seed culture of viruses egg embryo culture liquid; Cholera filtrate is provided by China national influenza center.

Two, experimental result and discussion

1, R1194 virus goes down to posterity: each mensuration for virus titer of R1194 sees Table 1:

Each mensuration of table 1:R1194 for virus titer

Method	P4	P5	P6	P7	P8	P9	P10
								Blood clotting titre (1/)	640	480	640	800	800	480	480
Virus titer (Lg)	8.5	8.0	8.0	7.5	7.67	7.5	8.5

2, the hypotype of R1194 seed culture of viruses is differentiated:

With A1, A3, B and H5N4 kind antiserum, intersect single immunodiffusion test with corresponding antigens, to differentiate its hypotype, result's (seeing Table 2) meets the H5 hypotype.

Table 2: various influenza virus intersection single immunodiffusion test

3, R1194 strain nucleotide sequencing:

1) the R1194 strain hemagglutinin nucleotide sequence that records is seen Fig. 1:

2) see Fig. 2 with original strain nucleotide sequence difference:

As can be seen from Figure 2: the nucleotide sequence of R1194 strain and original strain have relatively gone 12 bases of 1022 to 1033, are replaced by C at 1015,1034 and 1036 locational A.3) see Fig. 3 with original strain aminoacid sequence variation:

As can be seen from Figure 3: the R1194 strain is lacked four aminoacid of RRRK than original strain, and next K is replaced by T.The bird flu strain of the existing known R-R-R-K-K-R of containing aminoacid sequence belongs to the highly pathogenicity strain.

4, determining of inoculation titre, temperature and incubation time:

Table 3: the effect of different vaccination titre, temperature and incubation time virus relatively

Remarks: blood clotting titre (1/), virus titer (Lg)

Determine that cultivation temperature is 34.5 ℃, the inoculation titre is 10 ⁴, incubation time is 72h.

5, the calibrating of vaccine:

1) univalent vaccine stock solution verification result sees Table 4:

Table 4: univalent vaccine stock solution verification result

Type	Lot number	Liquid measure	Blood clotting content (μ g/mL)	Endotoxin (EU/mL)	Total protein (μ g/mL)	Total nitrogen/HA
							B/Malysia/2506/2004	B0701B	1000	281	<0.25	1028.0	3.66
NYMC?X-161B	A 3 0701B	1000	205	<0.25	257.2	1.25
							A/1VR-145	A 10701B	1000	331	<0.25	698.9	2.11
NIBRG-14	A 50701B	1000	90	<0.25	260.0	2.89

2) combined vaccine semi-finished product verification result sees Table 5:

Table 5: combined vaccine semi-finished product verification result

3) electron microscopic morphology before and after the R1194 virolysis is seen Fig. 4 and Fig. 5:

4) the SDS-PAGE electrophoresis result before and after the R1194 virolysis is seen Fig. 6:

As can be seen from Figure 6, before and after the purified virus cracking, all can see typical nucleic acid and hemagglutinin band.

5) immunogenicity inspection:

The third lactone inactivation of viruses after purification, the cracking, is made into 15 μ g/0.5ml, 4 of immunizing rabbits, and every intramuscular inoculation 0.5ml strengthens a pin after 15 days, and blood sampling in 30 days behind the first pin is measured blood and is pressed down antibody and neutralizing antibody, and the result is all greater than 1:40.

6) nucleic acid amplification of vaccine:

Provide the primer of making a definite diagnosis the H5N1 bird flu virus with national influenza center, the electrophoresis of amplification vaccine, the result amplifies the genetic fragment (see figure 7) of the bird flu virus of 378bp).This method also can be used for the discrimination test to this vaccine.

7, immunizing rabbit serum neutralizing antibody testing result interference phenomenon is analyzed:

1) the bird flu univalent vaccine of no adjuvant and human influenza-poultry influenza 4 valency combined vaccines are distinguished immunizing rabbit, relatively produce the situation (seeing Table 6) of avian influenza virus antibody:

Table 6: the titre that 2 seedlings of no adjuvant produce the bird flu virus neutralizing antibody compares (1/)

"-" expression＜1:10; The result: interference phenomenon is not obvious, and antibody titer is all greater than 1:20.

2) contain the bird flu univalent vaccine and the human influenza-poultry influenza 4 valency combined vaccines of adjuvant, immunizing rabbit respectively relatively produces the situation (seeing Table 7) of avian influenza virus antibody:

Table 7: the titre that contains the 2 seedlings generation bird flu virus neutralizing antibody of adjuvant compares (1/)

"-" expression＜1:10; The result: interference phenomenon is (P＜0.05) obviously, but the antibody of 1:76.9 is arranged.

3) the human influenza 3 valency vaccines of no adjuvant and people-bird flu 4 valency combined vaccines are distinguished immunizing rabbit, relatively produce the situation (seeing Table 8) of A1 type influenza virus neutralizing antibody:

Table 8: the titre that 2 seedlings of no adjuvant produce A1 type influenza virus neutralizing antibody compares (1/)

"-" expression＜1:10; The result: interference phenomenon one pin is not obvious; Two pins are (P＜0.05) obviously, but NAT is up to 1:615.1.

4) contain human influenza 3 valency vaccines and people-bird flu 4 valency combined vaccines of adjuvant, immunizing rabbit respectively relatively produces the situation (seeing Table 9) of A1 type influenza virus neutralizing antibody:

Table 9: the titre that contains the 2 seedlings generation A1 type influenza virus neutralizing antibody of adjuvant compares (1/)

"-" expression＜1:10; The result: interference phenomenon one pin is not obvious; Two pins are (P＜0.05) obviously, but NAT is up to 1:637.

5) the human influenza 3 valency vaccines of no adjuvant and people-bird flu 4 valency combined vaccines are distinguished immunizing rabbit, relatively produce the situation (seeing Table 10) of Type B influenza virus neutralizing antibody:

Table 10: the titre that 2 seedlings of no adjuvant produce Type B influenza virus neutralizing antibody compares (1/)

"-" expression＜1:10; Result: one pin＜1:10; Two pins are all very high, and interference phenomenon is not obvious.

6) contain human influenza 3 valency vaccines and people-bird flu 4 valency combined vaccines of adjuvant, immunizing rabbit respectively relatively produces the situation (seeing Table 11) of Type B influenza virus neutralizing antibody:

Table 11: the titre that contains the 2 seedlings generation Type B influenza virus neutralizing antibody of adjuvant compares (1/)

"-" expression＜1:10; Result: one pin＜1:10; Two pins are all very high, and interference phenomenon is not obvious.

7) the human influenza 3 valency vaccines of no adjuvant and people-bird flu 4 valency combined vaccines are distinguished immunizing rabbit, relatively produce the situation (seeing Table 12) of A3 type influenza virus neutralizing antibody:

Table 12: the titre that 2 seedlings of no adjuvant produce A3 type influenza virus neutralizing antibody compares (1/)

The result: interference phenomenon is not obvious; Titre is high after the 4 valency Seedling immunity, may be relevant with titre height before the immunity.

8) contain human influenza 3 valency vaccines and people-bird flu 4 valency combined vaccines of adjuvant, immunizing rabbit respectively relatively produces the situation (seeing Table 13) of A3 type influenza virus neutralizing antibody:

Table 13: the titre that contains the 2 seedlings generation A3 type influenza virus neutralizing antibody of adjuvant compares (1/)

The result: interference phenomenon is not obvious; Titre is high after the 4 valency Seedling immunity, may be relevant with titre height before the immunity.

9) people-bird flu 4 valencys are united no Adjuvanted vaccines, and immunizing rabbit 1 pin checked that the situation of neutralizing antibody sees Table 14 after 30 days:

Table 14: people-bird flu 4 valencys are united no adjuvant Seedling 30 days NAT of 1 pin (1/)

Rabbit number	A1	A3	B	H5N1
					1	540(—)	3480(640)	80(—)	5(—)
2	200(7.5)	6400(960)	100(—)	17.5(—)
					3	960(—)	8960(1280)	560(—)	60(—)
4	60(—)	2240(560)	40(—)	—(—)
					5	120(—)	8960(560)	50(—)	20(—)
Meansigma methods	236.9	5254.4(755.8)	89.1	10.1

Numeral in () is the neutralizing antibody before the immunity, "-" expression＜1:10; The result: only H5N1 is lower.

10) people-bird flu 4 valencys are united and are contained Adjuvanted vaccines, and immunizing rabbit 1 pin checked that the situation of neutralizing antibody sees Table 15 after 30 days:

Table 15: people-bird flu 4 valencys are united and are contained adjuvant Seedling 30 days NAT of 1 pin (1/)

Rabbit number	A1	A3	B	H5N1
					1	640(—)	12800(640)	480(—)	17.5(—)
2	1120(10)	17920(960)	1120(—)	320(—)
					3	640(—)	12800(1280)	640(—)	50(—)
4	1280(—)	1920(560)	400(—)	7.5(—)
					5	640(—)	3200(1120)	640(—)	35(—)
Meansigma methods	822.2	7099.7(868.2)	615.1	37.4

Numeral in () is the neutralizing antibody before the immunity, "-" expression＜1:10; Result: H5N1 is also greater than 1:20.

11) to press down the antibody test result basic similar to neutralizing antibody for rabbit immune serum blood.

12) the matched group rabbit is at experimental session, the same blood sampling simultaneously with experimental rabbit, and the result does not all detect influenza A1, Type B and H5N1 avian influenza antibody except that the A3 type influenza neutralizing antibody that detects similar titre, show that contrast sets up.

8, antibody test result sums up:

1) exempt from preceding rabbit antibody check result:

(1) do not find the neutralizing antibody of H 5 N 1 avian influenza and influenza Type B;

(2) detect the influenza A1 type neutralizing antibody that hangs down titre individually;

(3) very high influenza A3 type neutralizing antibody is generally arranged.

2) contain the inoculation of bird flu antigen vaccine:

(comprise bird flu epidemic disease unit price Seedling and people-bird flu unite 4 valency vaccines)

Behind 1 pin: no adjuvant or adjuvant Seedling all are not checked through neutralizing antibody.

Behind 2 pins: no adjuvant or adjuvant Seedling all are checked through neutralizing antibody, all〉1:20.

3) contain the inoculation of A type antigen vaccine:

(comprise human influenza 3 valency vaccines and people-bird flu unite 4 valency vaccines)

Behind 1 pin: no adjuvant or adjuvant Seedling, all check average〉the A type neutralizing antibody of 1:20;

Behind 2 pins: no adjuvant or adjuvant Seedling, all be checked through very high A type neutralizing antibody,

4) contain the inoculation of Type B antigen vaccine:

(comprise human influenza 3 valency vaccines and people-bird flu unite 4 valency vaccines)

Behind 1 pin: no adjuvant Seedling is not found neutralizing antibody; The adjuvant Seedling is found the neutralizing antibody of mean＜1:20;

Behind 2 pins: no adjuvant or adjuvant Seedling, all check higher neutralizing antibody.

5) contain the inoculation of A3 type antigen vaccine:

(comprise human influenza 3 valency vaccines and people-bird flu unite 4 valency vaccines)

Because before exempting from higher neutralizing antibody is arranged all, mean has reached about 1:800, behind 1 pin and 2 pins, neutralizing antibody is up to more than the 1:5000, thereby it is unnecessary to detect fowl stream A3 type neutralizing antibody.Having higher neutralizing antibody before rabbit is exempted from, may be that A3 type influenza virus is relevant with rabbit group wide-scale distribution the crowd, and 300 yuan every SPF rabbit from the Shanghai animal center is buied also detects very high neutralizing antibody before exempting from.

6) people-bird flu 4 valencys were united behind no adjuvant Seedling 1 pin 15 days, A1 type influenza virus neutralizing antibody has reached 1:49.2, and Type B neutralizing antibody＜1:10, but 30 days A1 types are higher behind 1 pin, reach 1:236.9, Type B also reaches 1:81.9, and hence one can see that, inoculates 1 pin people-bird flu, 4 valencys and unites no adjuvant Seedling, 3 kinds of viruses of A1, A3, Type B to human influenza are the same with traditional influenza vaccines, all can reach the excellent prevention effect, if make adjuvant Seedling, then better effects if.

7) detect the result that blood in the immunizing rabbit serum presses down antibody, similar to neutralizing antibody, because neutralizing antibody directly reflects the ability of virus killing, experimental result is stable, objective again, though and blood to press down the antibody method easy, the influence factor is more, reliability is not as neutralizing antibody.

9, as can be known: the associating of human influenza vaccine and avian influenza vaccine from The above results, substantially do not disturb, particularly do not have the adjuvant Seedling, even indivedual adjuvant Seedling is disturbed by some, but all produced higher neutralizing antibody, uniting of 2 kinds of vaccines of prompting is fully possible.

10, the equal acute safety testing by mice and Cavia porcellus of 6 seedlings.

11, the imagination of manufacturer-bird flu combined vaccine:

Technology: split vaccine-adult, child are suitable for.

Dosage form: no adjuvant Seedling-transparency liquid, with traditional influenza Seedling, easily promote; Or aluminium hydroxide adjuvant (0.5mg/0.5ml) vaccine, its immune effect is better.

Dosage: confirmer's influenza 1 pin of each 15 μ g-, bird flu 2 pins are effective.

Program: 1 pin is used to prevent human influenza; And, strengthen 1 pin when needing the burst birds flu-preventing again as the bird flu fundamental immunity.

2 pins are used for bird flu epidemic-stricken area resident, with the key population that contacts birds.

12, the same with influenza vaccines, along with the whole society annual constantly repeated inoculation and popularization, make the H5N1 avian influenza antibody in the crowd, constantly strengthen and enlarge, may reach the same like this with present H1N1 influenza, even H5N1 virus obtains the ability of human-to-human transmission, and is also not fearful.

Claims (10)

1. human influenza-poultry influenza combined vaccine, mainly comprise H1N1 type human influenza vaccine, H3N2 type human influenza vaccine, Type B human influenza vaccine and H5N1 type human and bird fluenza vaccine, each vaccine in hemagglutinin concentration is: H1N1 type human influenza vaccine 20～40 μ g/mL, H3N2 type human influenza vaccine 20～40 μ g/mL, Type B human influenza vaccine 20～40 μ g/mL and H5N1 type human and bird fluenza vaccine 20～40 μ g/mL.

2. human influenza-poultry influenza combined vaccine as claimed in claim 1 is characterized in that also containing in the described combined vaccine antiseptic phenoxyethanol 4～6mg/mL.

3. human influenza-poultry influenza combined vaccine as claimed in claim 1 is characterized in that also containing in the described combined vaccine final concentration and is 0.004%～0.007% tween 80.

4. human influenza-poultry influenza combined vaccine as claimed in claim 2 is characterized in that each vaccine in hemagglutinin concentration is: H1N1 type human influenza vaccine 30 μ g/mL, H3N2 type human influenza vaccine 30 μ g/mL, Type B human influenza vaccine 30 μ g/mL and H5N1 type human and bird fluenza vaccine 30 μ g/mL.

5. be that diluent is made as the described human influenza-poultry influenza combined vaccine of one of claim 1～4 with the phosphate buffer of 0.01mol/L, pH7.2.

6. as the described human influenza-poultry influenza combined vaccine of one of claim 1～4, it is characterized in that described H1N1 type human influenza vaccine is from human influenza H1N1 seed culture of viruses.

7. as the described human influenza-poultry influenza combined vaccine of one of claim 1～4, it is characterized in that described H3N2 type human influenza vaccine is from human influenza H3N2 seed culture of viruses.

8. as the described human influenza-poultry influenza combined vaccine of one of claim 1～4, it is characterized in that described Type B human influenza vaccine is from human influenza B seed culture of viruses.

9. as the described human influenza-poultry influenza combined vaccine of one of claim 1～4, it is characterized in that described H5N1 human and bird fluenza vaccine is from H 5 N 1 avian influenza attenuated strain seed culture of viruses.

10. the preparation method of human influenza-poultry influenza combined vaccine according to claim 1, described method comprises:

(1) human influenza H1N1/IVR-145 seed culture of viruses, human influenza H3N2/NYMC X-161B seed culture of viruses, human influenza B/Malysia-2506-2004 seed culture of viruses and H 5 N 1 avian influenza attenuated strain seed culture of viruses are produced univalent vaccine stock solution by following step respectively:

1. seed culture of viruses is inoculated in the chick embryo allantois intracavity after going down to posterity, diluting, and cultivates 48～72 hours for 33～35 ℃;

2. screen the live chickens embryo and put 2～8 ℃ of cold embryos 12～20 hours, draw the clarifying allantoic fluid of outward appearance and add formalin, put 2～8 ℃ and descended 18～20 hours, collect viral liquid, 40～50 times of ultrafiltration and concentration;

3. the viral liquid after concentrating adds β-third lactone that volume is a viral liquid long-pending 1/4000,2～8 ℃ of following deactivations 18～20 hours;

4. the viral liquid after the deactivation purified after, add final concentration and be 0.4% Triton X-100 or/and final concentration 0.4% NaTDC as decomposition agent, mix the back and handle 2～4min with the ultrasonic cell disruptor of 600W～700W power, put 20～30 ℃ again and act on 90～120 minutes down, obtain the virolysis thing;

5. the virolysis thing purified after, the tween 80 that adds volume and be lysate volume 0.006% behind the purification is as stabilizing agent, membrane filtration obtains univalent vaccine stock solution;

(2) will make univalent vaccine stock solution is mixed in proportion, phosphate buffer with 0.01mol/L, pH7.2 is diluted to H1N1 type human influenza vaccine concentration 20～40 μ g/mL, H3N2 type human influenza vaccine concentration 20～40 μ g/mL, Type B human influenza vaccine concentration 20～40 μ g/mL and H5N1 type human and bird fluenza vaccine concentration 20～40 μ g/mL by hemagglutinin content, and to add dense eventually be that the phenoxyethanol of 4～6mg/mL is as antiseptic, membrane filtration obtains described human influenza-poultry influenza combined vaccine.

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