CN101555281A

CN101555281A - Preparation method of anti-influenza A H1N1 virus specific IgY and related formulation thereof

Info

Publication number: CN101555281A
Application number: CNA2009101072241A
Authority: CN
Inventors: 包晟; 杨荣鉴; 包海威
Original assignee: SHENZHEN YACHEN BIOTECHNOLOGY TECHNOLOGY Co Ltd
Current assignee: SHENZHEN YACHEN BIOTECHNOLOGY TECHNOLOGY Co Ltd
Priority date: 2009-05-08
Filing date: 2009-05-08
Publication date: 2009-10-14

Abstract

The invention discloses a preparation method of an anti-influenza A H1N1 virus specific IgY and related formulations thereof. The method comprises the steps of preparing a plurality of types of complex antigens aiming at influenza A H1N1, using the complex antigens for immunizing laying hen, utilizing the immune egg yolk for preparing specific IgY crude extract dry powder, conducting purification, removing all types of bacterial virus by filtration, and then preparing the anti-variant influenza A H1N1 virus specific IgY, anti-influenza secondary infection bacterial specific IgY, or anti-variant influenza A H1N1 virus specific complex IgY. In addition, the invention makes the anti-variant influenza A H1N1 virus specific IgY into nanoliposomes, and the obtained nanoliposome anti-variant influenza A H1N1 virus specific IgY enhances healing efficacy. The anti-influenza A H1N1 virus specific IgY can be utilized for preparing various formulations which then can prevent and teat the influenza A H1N1 of human and animals fundamentally; and the invention is more convenient, more economic, safer and more effective than the control method used at the present time.

Description

The preparation method of anti-influenza A H 1 N 1 virus specific IgY and related preparations thereof

Technical field

The present invention relates to a kind of antibody and new formulation thereof, particularly relate to a kind of preparation method and related preparations thereof of anti-influenza A H 1 N 1 virus specific IgY.

Background technology

At first in Mexico's outburst, be diffused into world many countries and geographic influenza then rapidly and cause between in April, 2009 by a kind of H1N1 [A (H1N1) type] influenza virus.This H1N1virus is a human influenza virus, the North America avian influenza virus, and the mixture of North America, Europe and Asia swine influenza virus, human influenza virus, North America avian influenza virus have been comprised, and the gene composition of North America, Europe and Asia swine influenza virus, be a kind of new virus of producer sudden change.The most alarming is that this new virus is in person-to-person propagation.According to pig and human characteristics similar in blood relation, illustrate that both immunogenicities are similar with antigenicity.This just means that this new type influenza virus is the very fast adaptation of meeting in human body; Its epidemic strength can surpass SARS virus and avian influenza virus, and the mankind do not have immunizing power for this new virus.Therefore, the appearance of H1N1virus will constitute threat greatly to the mankind's life and health.Can in the process that infects the human and animal, constantly make a variation because H1N1virus is the same with other influenza virus, the immunity system of humans and animals is descended to its effect, reach the effect of " being braver after more fight "; Therefore, stoping this new type influenza viral prevalence, will be the secular challenge that the universe faces.

At present, the method that tackles Influenza A H1N1 mainly contains following several.

1, vaccination: vaccine is the effective means of flu-prevention, but the seasonal current influenza vaccine of whole world use at present is invalid to H1N1virus; China and some other country are all stepping up to develop the H1N1 vaccine now, estimate can come out soon.But because constantly drift and variation of the antigenicity of influenza virus, the new type influenza virus of this variation also can constantly make a variation certainly; And experience has in the past proved, and influenza virus often just makes a variation once half a year, and the development progress of influenza vaccines does not catch up with the influenza virus speed of mutation, therefore, has had a strong impact on the actual effect of influenza vaccines.Therefore, it is contemplated that the H1N1 vaccine that will come out is not as breaking through the different difficult problem of resistance, its destiny too.In addition, the H1N1 vaccine is the same with other influenza vaccines does not all have a therapeutic action.

2, isolate the infected: this is the method that the our times various countries generally adopt.Because the velocity of propagation of influenza virus is very fast, the infected has begun external toxin expelling when symptom not occurring; Therefore, in case be very popular, the tool border that isolates the infected acts as doubtful.

3, wear masks: because H1N1virus is very little with the general the same volume of influenza virus, general mouth mask can't stop H1N1virus at all.Therefore, the just psychology comfort that wears masks, actual do not have much effects.

4, port of entry temperature check: because the often also indefinite reveal any symptoms of influenza virus carrier more not necessarily can be had a fever when entry and exit; Therefore, can only chance on the sufferer who is having a fever individually, majority becomes " fish that has escape the net ".On April 30th, 2009, the flight from epidemic-stricken area Mexico to Shanghai obviously had a H1N1virus carrier, the temperature check on the airport, Shanghai through two hours on the machine; But, so tight multiple temperature check; But still fail to detect any unusual.This infected has just fallen ill behind Hong Kong as a result, becomes the first Influenza A H1N1 case of making a definite diagnosis within Chinese territory.

5, adopt neuraminidase inhibitors such as " Tamiflus ": evidence, zanamivir (Zanamivir) and Oseltamivir neuraminidase inhibitors such as (Oseltamivir) are to the H1N1virus sensitivity, therefore, this class medicine has certain therapeutic action to Influenza A H1N1.But such medicine has tangible central nervous system side effect, and Japan has occurred a lot ofly the case of mentally deranged symptom occurring because of taking " Tamiflu ".And clinical also proof is taken the propagation that this class medicine is prone to resistance poison pearl; Thereby, limited its application clinically.In addition, this medicine also has only certain therapeutic action, does not have preventive effect at all.

To sum up chatting, research and develop and a kind ofly can effectively prevent Influenza A H1N1, can effectively treat the medicine of new generation of Influenza A H1N1 again, just become the key subjects of pendulum before medical and health interface, countries in the world, also is the active demand of global public health service.

Summary of the invention

At the needs of prior art, the invention provides and a kind ofly can effectively prevent Influenza A H1N1, can effectively treat the medicine of new generation of Influenza A H1N1 again.

A kind of preparation method of anti-influenza A H 1 N 1 virus specific IgY is provided among the present invention, it is characterized in that, may further comprise the steps:

(S1) preparation is at the complex antigen of Influenza A H1N1;

(S2) get described complex antigen, bird inlay is carried out immunity, the hen of searching immunity produces immune egg;

(S3) get the yolk of described immune egg, stir, add distilled water, dilute and mix, make specific IgY crude extract dry powder again;

(S4) described crude extract dry powder is carried out purifying, various bacteriophagees of filtering again, make anti-influenza A H 1 N 1 virus specific IgY.

Complex antigen in the step of the present invention (S1) can comprise the influenza virus complex antigen, preparation process is as follows: get equivalent avian influenza vaccine liquid, porcine influenza vaccine liquid and seasonal human influenza vaccine liquid respectively, insert ultracentrifuge,, get supernatant with the 25000rpm high speed centrifugation; With conventional ammonium sulfate precipitation method precipitation virion, insert ultracentrifuge again, with the 25000rpm high speed centrifugation, get precipitation; Separate with Sepharose4B, collect avian influenza virus, swine influenza virus and seasonal human influenza virus's particle respectively; With three kinds of influenza viruses of sucrose density gradient ultracentrifugation method purifying, specifically be to get above-mentioned three kinds of influenza virus suspension 1.0ml that slightly carry respectively, be added to gently above the sucrose liquid of 40% and 60% discontinuous density gradient, through 100, centrifugal 2 hours of 000g gets sucrose layer intermediate zone band liquid, add pH7.2,0.01M phosphate buffered saline (PBS) 1.0ml, mixing, dialysis removes sugar, promptly gets the influenza virus suspension of three kinds of purifying; By 1: 1: 1 mixed, it was even to insert the homogenizer thorough mixing again with avian influenza virus, swine influenza virus and seasonal human influenza virus's suspension of described purifying, obtained containing the compound influenza virus mixed solution of three kinds of influenza virus compositions; The compound influenza virus mixed solution of getting above-mentioned preparation respectively adds 10-15% sodium lauryl sulphate (SDS), and cracking 30 minutes obtains the compound influenza antigen composition after the cracking; Add freund adjuvant in 1: 1 ratio, insert in the high speed homogenizer, with 8, the homogenize of 000rpm high speed, form water-in-oil liquid, promptly make the influenza virus complex antigen of three kinds of influenza virus cracking compositions such as containing avian influenza virus, swine influenza virus and seasonal human influenza virus;

Utilize this influenza virus complex antigen,, prepare the different influenza A H 1 N 1 virus specific IgY of resistance by described step (S2), (S3), (S4).

Complex antigen in the step of the present invention (S1) also can comprise three kinds of influenza virus polypeptide HA ₂Complex antigen, preparation process is as follows: use the RT-PCR method, respectively from H5N1 avian influenza virus, H3N2 swine influenza virus and seasonal H1N1 human influenza virus RNA clone hemagglutinin (HA ₂) remove the fragment of signal peptide and transmembrane domains in the gene; Insert the pGEM-T carrier earlier, after order-checking proved that the gene order that is obtained is correct, with restriction enzyme EcoR I and the two enzymic digestions of Not I, electrophoresis reclaimed the purpose fragment, is connected with the Yeast expression carrier pPIC9K that uses same two enzymic digestions; After transformed into escherichia coli, picking positive colony, upgrading grain, enzyme were cut and identified correctly, electricity transformed pichia yeast bacterium KM71 and GS115; Screening positive clone on the substratum that does not contain Histidine, and then the screening height copies transformant on the substratum of the G418 that contains different concns; The single colony inoculation of picking is in substratum, and in 28 degree shaking table overnight incubation, the dilution back continues to cultivate, and treats that the light absorption value that bacterial concentration reaches OD600 is about at 0.8 o'clock, changes substratum into contain methyl alcohol substratum, continues to cultivate 24-48 hour; In the different time sampling of cultivating, with the expression amount of HA in the ELISA method mensuration supernatant, select the highest time results of expression amount, centrifugal removal cell precipitation promptly contains the great expression product in the supernatant; Through 50% ammonium sulfate precipitation, the dialysis tubing of molecular weight cut-off 10kd was dialysed 24 hours with distilled water, and behind Sephacry1 S-200 and the Sephacry1 S-100 column chromatography, promptly obtained three kinds of influenza virus HA of purifying respectively ₂The antigen composition; With above-mentioned three kinds of influenza virus HA ₂The antigen composition expressing protein mixed by 1: 1: 1 earlier is even, add freund adjuvant in 1: 1 ratio again, insert high speed homogenizer, with 8, the homogenize of 000rpm high speed, form water-in-oil liquid, promptly make three kinds of influenza virus HA such as containing avian influenza virus, swine influenza virus and seasonal human influenza virus ₂The complex antigen of antigen composition expressing protein.

Utilize this complex antigen,, prepare the different influenza A H 1 N 1 virus specific IgY of resistance by described step (S2), (S3), (S4).

Complex antigen in the step of the present invention (S1) also can comprise Influenza A H1N1 secondary infection bacterium complex antigen, and preparation process is as follows: with A bunch of Type B Hemolytic streptococcus (2 * 10 ⁹/ ml), streptococcus pneumoniae (2 * 10 ⁹/ ml), hemophilus influenzae (2 * 10 ⁹/ ml) and streptococcus aureus (2 * 10 ⁹/ ml) the equivalent ratio is mixed with into the malignant bacteria mixture; With the freund adjuvant of this malignant bacteria mixture in 1: 1 ratio adding equivalent, with 8,000rpm handles, and becomes water in oil mixed bacterium body fluid, makes Influenza A H1N1 secondary infection bacterium complex antigen with high speed homogenizer again;

Utilize this Influenza A H1N1 secondary infection bacterium complex antigen,, prepare anti influenza secondary infection bacterium specific IgY by described step (S2), (S3), (S4).

The present invention also provides the preparation method of the different influenza A H 1 N 1 virus specific IgY of a kind of nanometer liposome resistance, it is characterized in that, may further comprise the steps:

Get the dry powder of the different influenza A H 1 N 1 virus specific IgY of resistance that described method makes, add pulverizings of milling in the supper micron mill, be processed into size between 1-100nm, granularity is above 〉=15000 purpose ultramicrons;

Yelkin TTS and cholesterol are dissolved in the ether, again described ultramicron is added the 4mmol/L phosphate buffered saline buffer and be made into IgY solution, supersound process 2min, the rotary evaporation that reduces pressure in water-bath immediately is to being gel, vortex oscillation makes the gel phase inversion, continues evaporation again and eliminates ether;

The ultramicron that does not wrap into is removed in the ultracentrifugation separation again, and precipitation washes secondary with water, and is centrifugal, must precipitate, and with 10mmol/L PBS dilution, promptly makes the different influenza A H 1 N 1 virus specific IgY of nanometer liposome resistance.

The present invention also provides a kind of resistance different Influenza A H1N1 specificity composite IgY, it is characterized in that, its composition comprises: different influenza A H 1 N 1 virus specific IgY of (1) aforementioned resistance or the different influenza A H 1 N 1 virus specific IgY of nanometer liposome resistance, (2) aforementioned anti influenza secondary infection bacterium specific IgY, above-mentioned two kinds of compositions are pressed (1～10): the mixed of (1～10) is even, promptly obtains the different Influenza A H1N1 specificity composite IgY of resistance.

The present invention also provides a kind of preparation at Influenza A H1N1, it is characterized in that, comprising in following four kinds of compositions one or more: the different influenza A H 1 N 1 virus specific IgY of (1) aforementioned resistance; (2) aforementioned anti influenza secondary infection bacterium specific IgY; (3) the different influenza A H 1 N 1 virus specific IgY of aforementioned nanometer liposome resistance; (4) the different Influenza A H1N1 specificity composite IgY of aforementioned resistance.

Among the present invention, described various preparations at Influenza A H1N1 also can comprise the compound composition that pharmaceutical chemicals and/or Chinese medicine are made into; Above-mentioned composition is equipped with distilled water, perhaps is equipped with various auxiliary materials, can be made into various human preparations, or makes for Mammals and bird use preparations such as pig, ox, sheep.

By the different influenza A H 1 N 1 virus specific IgY of resistance, anti influenza secondary infection bacterium specific IgY, the different influenza A H 1 N 1 virus specific IgY of nanometer liposome resistance and the different Influenza A H1N1 specificity composite IgY of resistance of method preparation of the present invention, the common pathogenic bacterium of Influenza A H1N1 secondary infection also there are good immunity conditioning and restraining effect; Therefore can be from eliminating viral infection and eliminating secondary infection two aspects and set about preventing in all directions and treat by the caused influenza of the H1N1virus of easy variation.This shows that this is more convenient, more economical, the safer more much effective again innovative product of a kind of product than existing widespread usage.

Description of drawings

The present invention does not have accompanying drawing.

Embodiment

IgY (Immunoglobulin of yolk) belongs to the IgG immunoglobulin like protein, effect with neutralizing antibody, it can combine with corresponding antigens generation specificity, thereby changes or suppressed the state or the activity of this antigen (as virus), stops this antigen (as virus) to be adsorbed in permissive cell; In addition, after its corresponding virus combination of IgY, form immunocomplex, easily engulfed by scavenger cell.

As previously mentioned, Influenza A H1N1 sense poison is the mixture of human influenza virus, North America avian influenza virus and North America, Europe and Asia swine influenza virus, has comprised the gene composition of human influenza virus, North America avian influenza virus and North America, Europe and Asia swine influenza virus; And its antigen is drift and variation constantly, and this has increased more difficulty for the development of anti-influenza A H 1 N 1 virus specific IgY.

The inventor the internal structure of further investigation Influenza A H1N1 sense poison and analyze this new virus comprehensively and the basis of the difference of common influenza virus on, adopt special method, succeed in developing a kind of specificity composite IgY that can effectively resist this three kinds of influenza virus mixtures, and use up-to-date nano lipsome body technique with this specificity composite IgY nanometer and liposomeization, make it easier, further improve curative effect by mucosal absorption; Simultaneously, adopt several different methods to solve the easily difficult problem of variation of Influenza A H1N1 sense poison.In addition, this special composite IgY also contains the antibody of anti influenza secondary infection bacterium, can effectively treat by Influenza A H1N1 sense poison and infect and the secondary infection that produces.It has overcome the H1N1 influenza virus to variation once more that " the Influenza A H1N1 vaccine " that be about to come out will run into not have the weakness that acts on, has remedied the shortcoming that the Influenza A H1N1 vaccine can not be used for the treatment of yet; More can replace countries in the world at present for stoping Influenza A H1N1 to propagate the ways and means of many " the getting half the result with twice the effort " of extensively adopting, become a kind of more convenient, more economical, safer more much effective again innovative product.

One, further investigation influenza virus structure

The contriver has thoroughly understood fully H1N1 influenza virus surface two peptide species antigens---the effect and the antigenic structure of hemagglutinin (HA) and neuraminidase (NA) by further investigation influenza virus structure, creatively utilizes HA ₂Between various and hypotype, belong to conservative protein, structure is more stable, variation is little, be again the characteristics of the protein ingredient that merges of mediation influenza virus coating and permissive cell film, research and develop continuous variation of a kind of H1N1 of solution influenza virus and the method for the problem that is difficult to tackle.

Two, preparation antigen composition

In view of the H1N1 influenza virus is human influenza virus, North America avian influenza virus, and the mixture of North America, Europe and Asia swine influenza virus, the present invention adopts two kinds of different methods to prepare the antigen composition, make these antigen compositions comprise human influenza virus, avian influenza virus, and the antigen composition of swine influenza virus.Purpose is to handle strains of influenza viruses with special method, exposes the HA of H1N1 strains of influenza viruses ₂Conserved regions improves its antigenicity, makes prepared IgY still effective to the H1N1 influenza virus after making a variation.The concrete grammar branch is chatted as follows:

(1) adopt the method for lytic virus strain to expose conserved regions composition in the virus, the compound influenza antigen composition after the acquisition cracking

1. separate three kinds of influenza viruses

(1) buys H5N1 avian influenza vaccine, H3N2 porcine influenza vaccine to Fujian Province's Sanming City herding poultry doctor station, buy seasonal human influenza trivalent vaccine to the Sanming City quarantine station.

(2) get equivalent avian influenza vaccine liquid, porcine influenza vaccine liquid and seasonal human influenza vaccine liquid respectively, insert ultracentrifuge,, get supernatant with the 25000rpm high speed centrifugation.Here be that to three kinds of vaccines respectively, independently handle following steps also are like this in (3) to (5).

(3) with conventional ammonium sulfate precipitation method precipitation virion, insert ultracentrifuge again,, get precipitation with the 25000rpm high speed centrifugation.

(4) separate with Sepharose 4B then, collect avian influenza virus, swine influenza virus and seasonal human influenza virus's particle respectively.

(5) with three kinds of influenza viruses of sucrose density gradient ultracentrifugation method purifying, concrete grammar is: get above-mentioned three kinds of influenza virus suspension 1.0ml that slightly carry respectively, be added to gently above the sucrose liquid of 40% and 60% discontinuous density gradient, through 100, centrifugal 2 hours of 000g gets sucrose layer intermediate zone band liquid, add pH7.2,0.01M phosphate buffered saline (PBS) 1.0ml, mixing, dialysis removes sugar, promptly gets three kinds of pure influenza virus suspension.

(6) with purified avian influenza virus, swine influenza virus and seasonal human influenza virus's suspension by 1: 1: 1 mixed, it is even to insert the homogenizer thorough mixing again, obtains containing the compound influenza virus mixed solution of three kinds of influenza virus compositions.

(7) handle compound influenza virus mixed solution with special method, expose the HA of three kinds of strains of influenza viruses ₂Conserved regions improves its antigenicity, makes prepared IgY still effective to the H1N1 influenza virus after making a variation.Concrete treatment process is: the compound influenza virus mixed solution of getting above-mentioned preparation respectively adds 10-15% sodium lauryl sulphate (SDS), and cracking 30 minutes promptly obtains the compound influenza antigen composition after the cracking; Through SDS-PAGE (sodium lauryl sulphate one polyacrylamide gel electrophoresis) check analysis, concentrated glue 4%, separation gel 7%, 240V, electrophoresis 30 minutes; Examine the dyeing of Ma Shi light blue, observe protein band; Detection determines to contain hemagglutinin heavy chain (HA ₁), hemagglutinin light chain (HA ₂), neuraminidase (NA), nucleoprotein (NP), P albumen (P ₁, P ₂, P ₃), stromatin (M ₁, M ₂) and Nonstructural Protein (NS, NS ₂).With Enami M, et al. experimental result unanimity.

(2) adopt three kinds of influenza virus polypeptide HA of genetic engineering technique reorganization ₂The antigen composition:

Clone hemagglutinin (HA from H5N1 avian influenza virus, H3N2 swine influenza virus and seasonal H1N1 human influenza virus RNA respectively with the RT-PCR method ₂) gene removes the fragment of signal peptide and transmembrane domains.Insert the pGEM-T carrier earlier.After order-checking proved that the gene order that is obtained is correct, with restriction enzyme EcoRI and the two enzymic digestions of Not I, electrophoresis reclaimed the purpose fragment, is connected with the Yeast expression carrier pPIC9K that uses same two enzymic digestions.Transformed into escherichia coli.After picking positive colony, upgrading grain, enzyme were cut and identified correctly, electricity transformed pichia yeast bacterium (Pichia pastoris) KM71 and GS115.Screening positive clone on the substratum that does not contain Histidine, and then the screening height copies transformant on the substratum of the G418 that contains different concns.The single colony inoculation of picking is in substratum, in 28 degree shaking table overnight incubation.The dilution back continues to cultivate.Treat that the light absorption value that bacterial concentration reaches OD600 is about at 0.8 o'clock, change substratum into contain methyl alcohol substratum, continue to cultivate 24-48 hour.In the different time sampling of cultivating, measure the expression amount of HA in the supernatant with the ELISA method.Select the highest time results of expression amount.Centrifugal removal cell precipitation.Promptly contain the great expression product in the supernatant.Through 50% ammonium sulfate precipitation, the dialysis tubing of molecular weight cut-off 10kd was dialysed 24 hours with distilled water, and behind Sephacry1 S-200 and the Sephacry1 S-100 column chromatography, promptly obtained three kinds of influenza virus HA of purifying respectively ₂The antigen composition.

Three, preparation influenza virus complex antigen

(1) complex virus is cracked into partitivirus antigen:

With the compound influenza antigen composition after the cracking of above-mentioned cleavage method preparation, in adding freund adjuvant in the viral mixed solution of 1: 1 ratio after cracking, insert in the high speed homogenizer, with 8, the homogenize of 000rpm high speed, form water-in-oil liquid, promptly make the viral complex antigen of three kinds of influenza virus cracking compositions such as containing avian influenza virus, swine influenza virus and seasonal human influenza virus.

(2) HA ₂Expressing protein antigen:

Three kinds of influenza virus HA with the obtained purifying of said gene engineering ₂Antigen composition expressing protein is earlier by even (100 micrograms/ml) of 1: 1: 1 mixed, then this mixed solution is added freund adjuvant in 1: 1 ratio, insert high speed homogenizer, with 8, the homogenize of 000rpm high speed, form water-in-oil liquid, promptly make three kinds of influenza virus HA such as containing avian influenza virus, swine influenza virus and seasonal human influenza virus ₂The antigen of antigen composition expressing protein.

Four, preparation Influenza A H1N1 secondary infection bacterium complex antigen

With A bunch of Type B Hemolytic streptococcus (2 * 10 ⁹/ ml), streptococcus pneumoniae (2 * 10 ⁹/ ml), hemophilus influenzae (2 * 10 ⁹/ ml) and streptococcus aureus (2 * 10 ⁹/ ml) the equivalent ratio is mixed with into the malignant bacteria mixture, with the freund adjuvant of this malignant bacteria mixture in 1: 1 ratio adding equivalent, with 8,000rpm handles with high speed homogenizer again, become water in oil mixed bacterium body fluid, make Influenza A H1N1 secondary infection bacterium complex antigen.

Five, the different H1N1virus immunity of preparation resistance egg

With virolysis composition complex antigen or the HA for preparing in above-mentioned the 3rd step ₂Antigen composition expressing protein antigen carries out immunity to bird inlay respectively; Strengthen injection more once every two weeks, planned immunization epidemic disease three times; Immunity for the first time is after 20 days, and the hen of searching immunity produces immune egg, and carries out coded markings.

Six, preparation anti influenza secondary infection bacterial immune egg

Influenza A H1N1 secondary infection bacterium complex antigen with preparing in above-mentioned the 4th step carries out immunity to bird inlay; Strengthen injection more once every two weeks, planned immunization epidemic disease three times, immunity for the first time is after 20 days, and the hen of searching after the immunity produces immune egg.The immune egg of being searched is carried out coded markings.

Above immunization method and frequency of injection can suitably be adjusted and variation according to hen immunne response situation, also can use above-mentioned same immunological technique, adopt above-mentioned not synantigen, respectively lay eggs duck or lay eggs female goose or the different egg birds such as the turkey or the ostrich of laying eggs of laying eggs are carried out immunity, obtain corresponding immune egg.

Seven, the preparation of crude extract

At first according to different and immune used antigen difference, with immune egg classification and label coding by the bird of immunity.Clean immune egg with flowing water, the wipes of alcohol wash disinfection, beating machiae is smashed immune egg, and egg white is removed in the sieving of yolk sieve, stays yolk, stirs; 4-6 by the egg yolk liquid volume extraordinarily goes into distilled water, dilutes and mixes, and transfers pH to 5.5-6.0 with 1.0N HCI solution.

The diluent of adjusting the pH value is further stirred, it is cooled to 2-6 ℃ then, left standstill 12 hours-24 hours; With diluent in 10, centrifugal 20 minutes of 000rpm; Getting separating obtained supernatant puts and carries out ultrafiltration and concentration 10-20 in the ultra-fine filter doubly; Then add sodium alginate solution, it is muddy to be stirred to appearance; Add 1.0%CaCl again ₂Liquid stirs, and 4 ℃ left standstill 8-12 hour; 8, centrifugal 20 minutes of 000rpm gets supernatant; The 0.22 μ m membrane filtration degerming 0.45 μ m film is contacted; Ultipor VF ^TMDV50 removes virus filter and removes virus removal; Lyophilize promptly makes the different influenza A H 1 N 1 virus specific IgY crude extract of resistance dry powder, anti influenza secondary infection bacterium specific IgY crude extract dry powder respectively; At last, prepared corresponding different antigenic specific IgY crude extract is carried out coded markings.

Eight, to the purifying of crude extract

Above three kinds of IgY crude extracts are dissolved in pH7.0,0.01M PB (phosphate buffered saline buffer) liquid respectively, successively cross ion exchange column and gel chromatography column chromatography more respectively, promptly make three kinds of pure product respectively, i.e. the pure product of the different influenza A H 1 N 1 virus specific IgY of resistance (dividing two kinds of different composite antigens), the pure product of anti influenza secondary infection bacterium specific IgY.

Nine, the various bacteriophagees of filtering

At the pure product of the 8th step gained, the bacteriophage filtering device that removes the virus filtration system that adopts U.S. Pall Ultrafine Filtration Company to make, thoroughly the various bacteriophagees of filtering guarantee that prepared IgY never contains any virus and bacterium.

The first road bacterium filtering device is to remove salmonella bacteriums such as (Salmonella) with 0.22 μ m film sterilizing filter; The second road mycoplasma filtering device is to remove the mycoplasma strainer with 0.1 μ m film to remove mycoplasma; The 3rd road virus filtering device is to use Ultipor VF ^TMDV50 removes virus filter and removes the multiple virus that comprises avian influenza virus, enterovirus.

Ten, the nanometer of the different influenza A H 1 N 1 virus specific IgY of resistance

Get the different influenza A H 1 N 1 virus specific IgY dry powder of the 9th prepared resistance of step, add the pulverizing of milling in the supper micron mill, be processed into size between 1-100nm, granularity surpasses 〉=15000 purpose ultramicrons, makes the different influenza A H 1 N 1 virus specific IgY dry powder of corresponding nanometer resistance.

11, the different influenza A H 1 N 1 virus specific IgY of resistance is made liposome liquid crystal micro-capsule

Yelkin TTS and cholesterol are dissolved in the ether, again the different influenza A H 1 N 1 virus specific IgY of nanometer resistance is added 4mmol/L phosphate buffered saline buffer (PBS) and be made into IgY solution, supersound process 2min (every processing 0.5min, .0.5min intermittently), rotary evaporation in water-bath, reduce pressure immediately to being gel, vortex oscillation makes the gel phase inversion, continue evaporation again and eliminate ether, and then ultracentrifugation (35000r/min, 30min) IgY that the end wraps into is removed in separation, and precipitation washes secondary with water, centrifugal, must precipitate,, promptly make the different influenza A H 1 N 1 virus specific IgY of nanometer liposome resistance with 10mmol/L PBS dilution.

The different influenza A H 1 N 1 virus specific IgY of above-mentioned various resistance, the different influenza A H 1 N 1 virus specific IgY of nanometer liposome resistance or anti influenza secondary infection bacterium specific IgY can add other pharmaceutical chemicals composition again or Chinese medicinal ingredients is made various compound medicines.

With the different influenza A H 1 N 1 virus specific IgY of above-mentioned various resistance, the different influenza A H 1 N 1 virus specific IgY of nanometer liposome resistance, anti influenza secondary infection bacterium specific IgY, perhaps their combination " the different Influenza A H1N1 specificity composite IgY of resistance ", add pharmaceutical chemicals, the compound composition that Chinese medicine etc. are made into is equipped with the solvent that distilled water is deployed into concentration 0.01-20.0%, can be made into various novel inhaling type propellants, nasal spray and mouth spraying agent (normal pressure or malleation), nasal drop, eye drops, throat spraying agent, formulation such as injection and air sanitizer.And the injection that contains the different Influenza A H1N1 specificity composite IgY of resistance is mainly used in emergency aid and treatment.

Also the different influenza A H 1 N 1 virus specific IgY of resistance, anti influenza secondary infection bacterium specific IgY can be pressed (1～10): the mixed of (1～10) is even, make a kind of " the different Influenza A H1N1 specificity composite IgY of resistance ", be equipped with the double solvents that distilled water is deployed into concentration 0.01-20.0%, make a kind of NEW TYPE OF COMPOSITE propellant or air sanitizer; Also the different influenza A H 1 N 1 virus specific IgY of nanometer liposome resistance the double solvents that distilled water is deployed into concentration 0.01-20.0% be can be equipped with, a kind of novel propellant or air sanitizer made.

With the different influenza A H 1 N 1 virus specific IgY of the resistance of 0.01-20.0%, or the different influenza A H 1 N 1 virus specific IgY of nanometer liposome resistance, perhaps " the different Influenza A H1N1 specificity composite IgY of resistance ", perhaps their combination, add chemical medicine, the compound composition that Chinese medicine is formed, be equipped with the auxiliary material of 99.99-80.0%, can be made into various clinical acceptable forms, as the inhaling type propellant, nasal spray and mouth spraying agent (normal pressure or malleation), nasal drop, eye drops, throat spraying agent, buccal tablet, air sanitizer, formulations such as injection are used for prevention and treatment Influenza A H1N1 and secondary infection.And the injection that contains the different influenza A H 1 N 1 virus specific composite IgY of resistance is mainly used in emergency aid and treatment.

In view of H1N1virus also is the main pathogens that causes pig and other animal breath tract disease, and at present the whole world prevent and treat that porcine influenza and other animal breath tract disease generally use medicine---microbiotic is not only like water off a duck's back to H1N1virus, instead can cause problems such as side effect, resistance and drug residue.In order more effectively to address this problem, can the anti-influenza A H 1 N 1 virus specific IgY of the present invention's research and development or composite IgY in addition solvent or various auxiliary material make novel animal with preparation for Mammals and bird uses such as pig, ox, sheep.But not only can effectively go out pig and the intravital H1N1virus of other animal, reach the effect of prevention and respiratory tract diseases such as treatment porcine influenza and other animal influenza; Can also block H1N1virus outwards distributes in pig body and other animal body.This Mammals and the bird preparations that use, that contain the different influenza A H 1 N 1 virus specific IgY of resistance such as pig, ox, sheep specialized in, can add various auxiliary materials and make pulvis, also can make aqua and injection, also can be made into a kind of natural safe air sanitizer and be used for place disinfections such as pig house, cowshed and diary farm and poulty house, duckery.

Applying nano liposome technology of the present invention is with the different influenza A H 1 N 1 virus specific IgY nanometer of prepared resistance, and super then microencapsulation increases penetrating power and prolongation of effect effect, improves actual efficacy.

The present invention utilizes the different H1N1virus immunoglobulin (Ig) of resistance (IgY) protection local mucous membrane (nasal cavity, the upper respiratory tract) cell the infecting of H1N1virus that do not made a variation; this is the key of its infection of blocking-up; sealing process is directly played in absorption to the variation H1N1virus, is that the different H1N1virus IgY of resistance has one of mechanism of prophylactic effect.The different H1N1virus IgY of resistance also can be infected the local new virus that discharges to the type A H1N1 influenza patient and be neutralized, and makes its forfeiture ability of propagating of row diffusion again, thereby reaches and not only can treat but also can prevent the dual purpose that Influenza A H1N1 is propagated.This is its innovation distinctive feature.

Just because of these advantages of IgY, the grippal purpose that is caused by H1N1virus is prevented and treated to the fatal shortcoming fairly obvious to the human body toxic side effect thereby reach with a definite target in view from the morbidity root to the actual killing action of H1N1virus is little just can to overcome antiviral.

Because HA ₂Be the albumen composition that the corpusculum film merges in mediation influenza virus film and the cell, the present invention makes a kind of anti-influenza A H 1 N 1 virus HA ₂IgY, this anti-influenza A H 1 N 1 virus HA ₂Antibody can stop that the corpusculum film merges in H1N1virus coating and the cell, and even influence H1N1virus and cytolemma fusion, cause the virus nucleocapsid body can not enter in the cell; Thereby, H1N1virus is produced the effect of effectively preventing.Like this, even the variable region (heavy chain-HA of H1N1virus ₁) variation has taken place, as previously mentioned, its conserved regions (light chain-HA ₂) be constant; Adopt the specific IgY or the anti-influenza A H 1 N 1 virus HA of each polypeptide antigen of the different H1N1virus of resistance (comprising the conserved regions that exposes after the cracking) of method preparation of the present invention ₂The specific IgY of expressing protein still can produce the effect of effectively checking to it.

Following experimental example and embodiment are used to further specify the present invention.

Experimental example 1: the content detection of different influenza A H 1 N 1 virus specific IgY of resistance and anti influenza secondary infection bacterium specific IgY

Use SDS-PAGE (sodium lauryl sulphate one polyacrylate hydrogel) cataphoretic determination method, respectively the different influenza A H 1 N 1 virus specific IgY of different resistances, the anti influenza secondary infection bacterium specific IgY crude extract of producing by above technology detected, the result contains IgY 45-52%.The IgY crude extract obtains pure IgY after secondary is crossed post.Analyze through SDS-PAGE, it is pure that purity reaches PAGE, as shown in the table:

The pure product of IgY	Pure IgY content
The pure product of IgY	Pure IgY content	The different influenza A H 1 N 1 virus specific IgY of resistance	99.99％
Anti influenza secondary infection bacterium specific IgY	99.99％		99.99％

Experimental example 2: the activity of different influenza A H 1 N 1 virus specific IgY of resistance and anti influenza secondary infection bacterium specific IgY detects

Adopt the prepared resistance of the Technology different H1N1 influenza virus specific IgY and the anti influenza secondary infection bacterium specific IgY of inventor's invention, adopt " ELISA method " (enzyme linked immunosorbent assay) to carry out activity and detect.

Detected result shows, the prepared two kinds of different H1N1 influenza virus of different resistances specific IgYs, and wherein the I type is tired to the antibodies of H1N1virus and has been reached more than 1: 1024.And the II type is tired to the antibodies of H1N1virus and is also reached more than 1: 512.Equally, anti influenza secondary infection bacterium specific IgY is to representational pneumococcus, A bunch Type B haemolysis type suis, streptococcus aureus, and the antibody titer of hemophilus influenzae is also all more than 1: 256.

Two kinds of different influenza A H 1 N 1 virus specific IgYs of resistance detected result of tiring

The anti-secondary infection bacterium specific IgY detected result of tiring

Experimental example 3: with the genetically engineered HA that recombinates ₂Expressing protein prepares anti-influenza A H 1 N 1 virus HA ₂The activity of specificity chicken IgY detects

Adopt described genetically engineered reorganization H1N1virus polypeptide method of the present invention to make HA ₂The pure product of expressing protein.

With this H1N1virus HA ₂Expressing protein 1.0mg adds freund adjuvant in 1: 1 ratio, and inserts the homogenize of high speed homogenizer high speed, and preparation contains H1N1virus HA ₂The antigen of expressing protein.

Adopt method of the present invention with this H1N1virus HA that contains ₂The antigen immune bird inlay of expressing protein prepares anti-influenza A H 1 N 1 virus HA ₂Specific IgY.Again respectively with the seasonal human influenza virus of H1N1virus and H1N1 as detecting antigen, detect this anti-influenza A H 1 N 1 virus HA with " ELISA " method ₂Specific IgY is to these two kinds of antigenic antibody titers of difference.The result is as follows:

Above result shows, adopts genetically engineered to prepare H1N1virus HA ₂Antigen, prepared anti-influenza A H 1 N 1 virus HA ₂Specific IgY all includes for inside configuration and has conservative property HA ₂The seasonal human influenza virus of the H1N1virus of polypeptide and H1N1 has all produced immunological response, certain tiring arranged, this shows that this IgY has restraining effect to these two kinds of subtype influenza viruses, has verified that the prepared IgY of technology of the present invention has the different characteristic of resistance.

Experimental example 4: detect as the activity that complex antigen prepares the different influenza A H 1 N 1 virus specific duck of resistance IgY with the virolysis composition

Buy " seasonal human influenza trivalent vaccine " 200 to Sanming City, Fujian quarantine station, buy " porcine influenza vaccine " and " avian influenza vaccine " each 200 to Sanming City herding poultry doctor station; With these three kinds of vaccines by after preceding chatting the method isolated viral and purifying, obtain compound influenza virus mixed solution after mixing evenly in 1: 1: 1 ratio again, adopt aforementioned " virolysis method " to make lysate then, at last this lysate is added freund adjuvant in 1: 1 ratio, and insert the homogenize of high speed homogenizer high speed, preparation contains the viral complex antigen of three kinds of influenza virus cracking compositions such as avian influenza virus, swine influenza virus and seasonal human influenza virus.

Adopt method of the present invention with this viral complex antigen immunity duck of laying eggs, prepare the different influenza A H 1 N 1 virus specific duck of resistance IgY.Again respectively with H1N1virus and H9N2 avian influenza virus and H3N2 swine influenza virus as detecting antigen, detect the different influenza A H 1 N 1 virus specific duck of this resistance IgY with " ELISA " method these three kinds of antigenic antibodies of difference tired.The result is as follows:

Test-results shows that employing virolysis legal system of the present invention is equipped with lytic virus antigen immune bird, the different influenza A H 1 N 1 virus specific IgY of prepared resistance not only has very strong inhibition activity (antibodies is tired) to the human influenza virus, avian influenza virus and swine influenza virus also all there are very strong inhibition activity (antibodies is tired), have verified that further the prepared IgY of technology of the present invention has the different characteristic of ideal resistance; Also explanation simultaneously: the different influenza A H 1 N 1 virus specific IgY of this resistance also can be used for preventing and treating bird flu and influenzas such as pig, ox except can be used for human prevention and treatment influenza.

Experimental example 5: the activity of anti influenza secondary infection specificity chicken composite IgY detects

Adopt ordinary method to cultivate A bunch of Type B Hemolytic streptococcus, streptococcus pneumoniae, hemophilus influenzae, streptococcus aureus the contriver in the biochemical test chamber of high and new technology industrial development zone, Shenzhen.Then with above 4 kinds of pathogenic bacterium by 1: 1: 1: 0.5 mixed, make 10ml bacterium liquid and add the 10ml freund adjuvant again and insert the homogenize of high speed homogenizer high speed then, make influenza secondary infection bacterium complex antigen 20ml.

Adopt this complex antigen immune hen that contains full bacterium, preparation anti influenza secondary infection specific IgY.Then with the different influenza A H 1 N 1 virus specific IgY of any one resistance (the virolysis antigen or the HAHA of itself and method for preparing ₂Expressing protein antigen) mixed by 1: 1 is even, makes different Influenza A H1N1 of resistance and secondary infection specificity composite IgY.

At last, respectively with the seasonal human influenza virus of H1N1virus, H9N2 avian influenza virus, H3N2 and A bunch of Type B Hemolytic streptococcus, streptococcus pneumoniae, hemophilus influenzae, streptococcus aureus as detecting antigen, detect this composite IgY to these antigenic antibody titers with " ELISA " method, the result is as follows:

Show that from above detected result by prepared different Influenza A H1N1 of resistance and the secondary infection specificity composite IgY of method of the present invention, to three different subtype influenza viruses, and common several secondary infection pathogenic bacterium all have higher tiring.

Embodiment 1: be equipped with special chicken IgY of the different H1N1virus of resistance with the virolysis legal system

1. separate and three kinds of influenza viruses of purifying

(2) get equivalent avian influenza vaccine liquid, porcine influenza vaccine liquid and seasonal human influenza vaccine liquid respectively, insert ultracentrifuge,, get supernatant with the 25000rpm high speed centrifugation.

(4) separate with Sepharose 4B then, collect avian influenza virus and swine influenza virus and seasonal human influenza virus's particle respectively.

(5) with three kinds of influenza viruses of sucrose density gradient ultracentrifugation method difference purifying, concrete grammar is as follows:

Get above-mentioned three kinds of influenza virus suspension 1.0ml that slightly carry respectively, be added to gently above the sucrose liquid of 40% and 60% discontinuous density gradient, through 100, centrifugal 2 hours of 000g, get sucrose layer intermediate zone band liquid, add pH7.2,0.01M phosphate buffered saline (PBS) 1.0ml, mixing, dialysis removes sugar, promptly gets three kinds of pure influenza virus suspension.

2. prepare compound influenza virus mixed solution

With purified avian influenza virus suspension and swine influenza virus suspension and seasonal human influenza virus's suspension by 1: 1: 1 mixed, it is even to insert the homogenizer thorough mixing again, obtains containing the compound influenza virus mixed solution of three kinds of influenza virus compositions.

3. lytic virus

Handle compound influenza virus mixed solution with special method, expose the HA2 conserved regions of three kinds of strains of influenza viruses, improve its antigenicity, make prepared IgY still effective the H1N1 influenza virus after making a variation.Concrete treatment process is as follows:

The compound influenza virus mixed solution of getting above-mentioned preparation respectively adds 10-15% sodium lauryl sulphate (SDS), and cracking 30 minutes promptly obtains the compound influenza antigen composition after the cracking.Through SDS-PAGE (sodium lauryl sulphate one polyacrylamide gel electrophoresis) check analysis, concentrate glue 4%, separation gel 7%, 240V, electrophoresis 30 minutes.Examine the dyeing of Ma Shi light blue, observe protein band.Detection determines to contain hemagglutinin heavy chain (HA ₁), hemagglutinin light chain (HA ₂), neuraminidase (NA), nucleoprotein (NP), P albumen (P ₁, P ₂, P ₃), stromatin (M ₁, M ₂) and Nonstructural Protein (NS, NS ₂).With Enami M, et al. experimental result unanimity.

4. the preparation complex virus is cracked into partitivirus antigen

With the compound influenza antigen composition after the cracking of the above-mentioned cleavage method preparation of the present invention, in adding freund adjuvant in the viral mixed solution of 1: 1 ratio after cracking, insert in the high speed homogenizer, with 8, the homogenize of 000rpm high speed, form water-in-oil liquid, promptly make the viral complex antigen of three kinds of influenza virus cracking compositions such as containing avian influenza virus, swine influenza virus and seasonal human influenza virus, meter 150ml.

5. prepare the different H1N1virus immunity of resistance egg

With 50 of the bird inlays of the viral complex antigen immune health of this influenza virus cracking composition, every intramuscularly 0.5ml every the injection of two weeks once, amounts to three times.

6. prepare the different influenza A H 1 N 1 virus specific IgY crude extract of resistance

Clean immune egg with flowing water, the wipes of alcohol wash disinfection, beating machiae is smashed immune egg, and egg white is removed in the sieving of yolk sieve, stays yolk, stirs; 4-6 by the egg yolk liquid volume extraordinarily goes into distilled water, dilutes and mixes, and transfers pH to 5.5-6.0 with 1.0N HCI solution.

The diluent of adjusting the pH value is further stirred, it is cooled to 2-6 ℃ then, left standstill 12 hours-24 hours; With diluent in 10, centrifugal 20 minutes of 000rpm; Getting separating obtained supernatant puts and carries out ultrafiltration and concentration 10-20 in the ultra-fine filter doubly; Then add sodium alginate solution, it is muddy to be stirred to appearance; Add 1.0%CaCl again ₂Liquid stirs, and 4 ℃ left standstill 8-12 hour; 8, centrifugal 20 minutes of 000rpm gets supernatant; The 0.22 μ m membrane filtration degerming 0.45 μ m film is contacted; Ultipor VF ^TMDV50 removes virus filter and removes virus removal; Lyophilize promptly makes the different influenza A H 1 N 1 virus specific chicken of resistance IgY crude extract dry powder 750 grams.

7. the different influenza A H 1 N 1 virus specific IgY of purifying resistance

The different influenza A H 1 N 1 virus specific chicken of above resistance IgY crude extract is dissolved in pH7.0,0.01M PB (phosphate buffered saline buffer) liquid, successively cross ion exchange column and gel chromatography column chromatography more respectively, then make the pure product of the different influenza A H 1 N 1 virus specific chicken of resistance IgY.

8. the bacteriophage filtering device that removes the virus filtration system that adopts U.S. Pall Ultrafine Filtration Company to make, thoroughly the various bacteriophagees of filtering guarantee that prepared IgY never contains any virus and bacterium.

Embodiment 2: the different influenza A H 1 N 1 virus specific IgY nanometer of resistance

Get the different influenza A H 1 N 1 virus specific chicken of prepared resistance IgY dry powder, add the pulverizing of milling in the supper micron mill, be processed into size between 1-100nm, granularity surpasses 〉=15000 purpose ultramicrons, makes the different influenza A H 1 N 1 virus specific chicken of corresponding nanometer resistance IgY dry powder.

Embodiment 3: the different influenza A H 1 N 1 virus specific IgY of resistance is made liposome liquid crystal micro-capsule.

Yelkin TTS and cholesterol are dissolved in the ether, again the different influenza A H 1 N 1 virus specific IgY of nanometer resistance is added 4mmol/L phosphate buffered saline buffer (PBS) and be made into IgY solution, supersound process 2min (every processing 0.5min, .0.5min intermittently), rotary evaporation in water-bath, reduce pressure immediately to being gel, vortex oscillation makes the gel phase inversion, continue evaporation again and eliminate ether, and then ultracentrifugation (35000r/min, 30min) IgY that does not wrap into is removed in separation, and precipitation washes secondary with water, centrifugal, must precipitate,, promptly make the different influenza A H 1 N 1 virus specific chicken of resistance IgY liposome with 10mmol/L PBS dilution.

Embodiment 4: be equipped with resisiting influenza virus specificity goose IgY with the virolysis legal system

1. separate and three kinds of influenza viruses of purifying

(5) with three kinds of influenza viruses of sucrose density gradient ultracentrifugation method purifying, concrete grammar is as follows:

2. prepare compound influenza virus mixed solution

3. lytic virus

Handle compound influenza virus mixed solution with special method, expose the HA of three kinds of strains of influenza viruses ₂Conserved regions improves its antigenicity, makes prepared IgY still effective to the H1N1 influenza virus after making a variation.Concrete treatment process is as follows:

4. the preparation complex virus is cracked into partitivirus antigen

With the compound influenza antigen composition after the cracking of the above-mentioned cleavage method preparation of the present invention, in adding freund adjuvant in the viral mixed solution of 1: 1 ratio after cracking, insert in the high speed homogenizer, with 8, the homogenize of 000rpm high speed forms water-in-oil liquid, promptly makes the viral complex antigen of three kinds of influenza virus cracking compositions such as containing avian influenza virus, swine influenza virus and seasonal human influenza virus, meter 2,250ml.

5. prepare the different H1N1virus immunity of resistance egg

Adopt 100 female gooses of breeding of eliminating the high immunne response ability of the preferred tool of method earlier, adopt the above-mentioned viral complex antigen that contains three kinds of influenza virus cracking compositions, meter 1,500ml carries out immunity to female goose respectively, per injection 5ml antigen, after injection for the first time, strengthen injection more once every two weeks, totally three times, in injecting the back seven month for the first time, the IgY that extracts respectively according to a conventional method behind the egg mark that these geese produced wherein, detect tiring of prepared IgY with ELISA method (enzyme linked immunosorbent assay), select and wherein can make IgY and tire 〉=256 the strong especially goose of immunne response ability, the egg that is produced with this batch goose is hatched good goose kind again, treat that it grows up to 2-3 month, select the wherein extraordinary female goose of 50 preferred high immunne response abilities of conduct.

Then, with 50 of the female gooses of the high immunne response that optimizes of the viral complex antigen immune health of this influenza virus cracking composition, adopt intravenous reinforced immunological method, respectively preferred extraordinary female goose is carried out immunity, every female goose per injection amount reaches 5ml antigen, strengthen injection more once every two weeks, be total to immunity three times in one month; Immunity was each time searched female goose and is produced immune egg to the 7 months termination after 20 days, examine altogether 7,500 on immune goose egg.

6. prepare the different influenza A H 1 N 1 virus specific goose of resistance IgY crude extract

Clean immune goose egg with flowing water, the wipes of alcohol wash disinfection, beating machiae is smashed immune goose egg, and egg white is removed in the sieving of yolk sieve, stays yolk, stirs; 4-6 by the egg yolk liquid volume extraordinarily goes into distilled water, dilutes and mixes, and transfers pH to 5.5-6.0 with 1.0N HCI solution.

The diluent of adjusting the pH value is further stirred, it is cooled to 2-6 ℃ then, left standstill 12 hours-24 hours; With diluent in 10, centrifugal 20 minutes of 000rpm; Getting separating obtained supernatant puts and carries out ultrafiltration and concentration 10-20 in the ultra-fine filter doubly; Then add sodium alginate solution, it is muddy to be stirred to appearance; Add 1.0%CaCl again ₂Liquid stirs, and 4 ℃ left standstill 8-12 hour; 8, centrifugal 20 minutes of 000rpm gets supernatant; The 0.22 μ m membrane filtration degerming 0.45 μ m film is contacted; Ultipor VF ^TMDV50 removes virus filter and removes virus removal; Lyophilize promptly makes the different influenza A H 1 N 1 virus specific goose of resistance IgY crude extract dry powder meter 2,200 grams.

7. the different influenza A H 1 N 1 virus specific goose of purifying resistance IgY

The different influenza A H 1 N 1 virus specific chicken of above resistance IgY crude extract is dissolved in pH7.0,0.01M PB (phosphate buffered saline buffer) liquid, successively cross ion exchange column and gel chromatography column chromatography more respectively, then make pure product 400 grams of the different influenza A H 1 N 1 virus specific goose of resistance IgY.

Embodiment 5: prepare anti-HA with gene engineering research ₂Specificity chicken IgY

1. adopt three kinds of influenza virus polypeptide HA of genetic engineering technique reorganization ₂The antigen composition

2. prepare HA ₂Expressing protein antigen

Three kinds of influenza virus HA with the obtained purifying of said gene engineering of the present invention ₂Antigen composition expressing protein is by even (100 micrograms/ml) of 1: 1: 1 mixed, add freund adjuvant in 1: 1 ratio again, insert high speed homogenizer, with 8, the homogenize of 000rpm high speed, form water-in-oil liquid, promptly make three kinds of influenza virus HA such as containing avian influenza virus, swine influenza virus and seasonal human influenza virus ₂The antigen of antigen composition expressing protein, meter 300ml.

3. prepare the different H1N1virus immunity of resistance egg

With this three kinds of influenza virus HA ₂100 of the bird inlays of the antigen immune health of antigen composition expressing protein, every intramuscularly 1ml every the injection of two weeks once, amounts to three times.

4. prepare the different influenza A H 1 N 1 virus specific chicken of resistance IgY crude extract

The diluent of adjusting the pH value is further stirred, it is cooled to 2-6 ℃ then, left standstill 12 hours-24 hours; With diluent in 10, centrifugal 20 minutes of 000rpm; Getting separating obtained supernatant puts and carries out ultrafiltration and concentration 10-20 in the ultra-fine filter doubly; Then add sodium alginate solution, it is muddy to be stirred to appearance; Add 1.0%CaCl again ₂Liquid stirs, and 4 ℃ left standstill 8-12 hour; 8, centrifugal 20 minutes of 000rpm gets supernatant; The 0.22 μ m membrane filtration degerming 0.45 μ m film is contacted; Ultipor VF ^TMDV50 removes virus filter and removes virus removal; Lyophilize promptly makes the different influenza A H 1 N 1 virus specific chicken of resistance IgY crude extract dry powder 1500 grams.

5. the different influenza A H 1 N 1 virus specific IgY of purifying resistance

The different influenza A H 1 N 1 virus specific chicken of above resistance IgY crude extract is dissolved in pH7.0,0.01M PB (phosphate buffered saline buffer) liquid, successively cross ion exchange column and gel chromatography column chromatography more respectively, then make the pure product of the different influenza A H 1 N 1 virus specific chicken of resistance IgY, meter 300 grams.

6. the bacteriophage filtering device that removes the virus filtration system that adopts U.S. Pall Ultrafine Filtration Company to make, thoroughly the various bacteriophagees of filtering guarantee that prepared IgY never contains any virus and bacterium.

Embodiment 6: preparation anti influenza secondary infection specific IgY

(1) preparation influenza secondary infection bacterium complex antigen

With A bunch of Type B Hemolytic streptococcus (2 * 10 ⁹/ ml), streptococcus pneumoniae (2 * 10 ⁹/ ml), hemophilus influenzae (2 * 10 ⁹/ ml) and streptococcus aureus (2 * 10 ⁹/ ml) the equivalent ratio is mixed with into the malignant bacteria mixture, again with the freund adjuvant of malignant bacteria mixture in 1: 1 ratio adding equivalent, with 8,000rpm handles with high speed homogenizer, and promptly getting water in oil mixed bacterium isoantigen is secondary infection malignant bacteria complex antigen.

(2) preparation anti influenza secondary infection bacterial immune egg

With prepared influenza secondary infection bacterium complex antigen, 5 of immune bird inlays; Every intramuscular injection 0.5ml.Strengthen injection more once every two weeks, planned immunization epidemic disease three times, immunity for the first time is after 20 days, and the hen of searching after the immunity produces immune egg, to 7th month, searches 7,500 immune eggs altogether.

(3) preparation anti influenza secondary infection bacterium specific IgY crude extract

The immune egg of being searched is cleaned with flowing water, and the wipes of alcohol wash disinfection is smashed immune egg with beating machiae again, removes egg white with the sieving of yolk sieve, stays yolk, stirs; 4-6 by the egg yolk liquid volume extraordinarily goes into distilled water, dilutes and mixes, and transfers pH to 5.5-6.0 with 1.0N HCI solution.

The diluent of adjusting the pH value is further stirred, it is cooled to 2-6 ℃ then, left standstill 12 hours-24 hours; With diluent in 10, centrifugal 20 minutes of 000rpm; Getting separating obtained supernatant puts and carries out ultrafiltration and concentration 10-20 in the ultra-fine filter doubly; Then add sodium alginate solution, it is muddy to be stirred to appearance; Add 1.0%CaCl again ₂Liquid stirs, and 4 ℃ left standstill 8-12 hour; 8, centrifugal 20 minutes of 000rpm gets supernatant; The 0.22 μ m membrane filtration degerming 0.45 μ m film is contacted; Ultipor VF ^TMDV50 removes virus filter and removes virus removal; Lyophilize promptly makes anti influenza secondary infection bacterium specific IgY crude extract dry powder, meter 700 grams.

(4) purifying of anti influenza secondary infection bacterium specific IgY:

Prepared anti influenza secondary infection bacterium specific IgY crude extract is dissolved in pH7.0,0.01MPB (phosphate buffered saline buffer) liquid, successively cross ion exchange column and gel chromatography column chromatography more respectively, promptly make pure product 150 grams of anti influenza secondary infection bacterium specific IgY.

Embodiment 7: the agent of the different influenza A H 1 N 1 virus specific IgY normal pressure spray of preparation resistance

Adopt the different influenza A H 1 N 1 virus specific IgY 50g of resistance, make the agent of a kind of anti-H 1 N 1 influenza normal pressure spray as main raw material.

The different influenza A H 1 N 1 virus specific IgY 50g of resistance

Glycerine 1,000g

PEG400 1,250g

Mentha camphor 50g

Essence 15g

Ethanol 500g (500ml)

Distilled water adds to 50kg (50 liters)

The different influenza A H 1 N 1 virus specific IgY of resistance is dissolved in the distilled water, adds the glycerine mixing.Mentha camphor, essence add dissolve with ethanol, add the PEG400 mixing again, under agitation are added on above-mentioned solution and stir evenly, filter, and add distilled water to full dose, transfer pH to 6.5 with 0.1mol, NaOH flow liquid.Soup is poured in the normal pressure spray bottle, and every 20ml examines qualified back packing entirely and promptly gets 2,500 anchorage Influenza A H1N1 IgY normal pressure spray agent.

Embodiment 8: preparation nanometer liposome anti-influenza A H 1 N 1 virus specific IgY nasal spray

Adopt the nanometer liposome different influenza A H 1 N 1 virus specific IgY 1000ml of resistance (1000g), make a kind of anti-H 1 N 1 influenza nasal spray as main raw material.

The different influenza A H 1 N 1 virus specific IgY 1 of nanometer liposome resistance, and 000g (1,000ml)

Glycerine 20,000g

PEG400 25,000g

Mentha camphor 1000g

Essence 300g

Ethanol 1, and 000g (1,000ml)

Distilled water adds to 1000kg (1000 liters)

The different influenza A H 1 N 1 virus specific IgY of nanometer liposome resistance is dissolved in the distilled water, adds the glycerine mixing.Mentha camphor, essence add dissolve with ethanol, add the PEG400 mixing again, under agitation are added on above-mentioned solution and stir evenly, filter, and add distilled water to full dose, transfer pH to 6.5 with 0.1mol, NaOH flow liquid.Soup is poured in the normal pressure spray bottle, and every 20ml examines qualified back packing entirely and promptly gets 50,000 liposome anti-H 1 N 1 influenza IgY nasal sprays.

Embodiment 9: prepare the agent of a kind of compound anti-H 1 N 1 influenza normal pressure spray

With becoming anti-H 1 N 1 influenza specificity composite IgY 200g (anti-influenza A H 1 N 1 virus specific IgY 133.33g, anti influenza secondary specific IgY 66.66g) with anti influenza secondary infection specific IgY by 2: 1 mixed with the 6 prepared anti-influenza A H 1 N 1 virus specific IgYs of method that describe in detail, make the agent of a kind of compound anti-H 1 N 1 influenza normal pressure spray as main raw material with this by embodiment 1.

Anti-H 1 N 1 influenza specificity composite IgY 200g

Glycerine 4,000g

PEG400 5,000g

Mentha camphor 200g

Essence 80g

Ethanol 2, and 000g (2,000ml)

Distilled water adds to 200kg (200 liters)

The anti influenza specificity composite IgY is dissolved in 160 liters of distilled water, adds the glycerine mixing.Mentha camphor, essence add dissolve with ethanol, add the PEG400 mixing again, under agitation are added on above-mentioned solution and stir evenly, filter, and add distilled water to full dose, transfer pH to 7.0, can with 0.1mol, NaOH solution.Soup is poured in the normal pressure spray bottle, and every 20ml examines qualified back packing entirely and promptly gets the agent of 10,000 compound anti-H 1 N 1 influenza IgY normal pressure sprays.

Embodiment 10: preparation anti-H 1 N 1 influenza sterilization sprays with fresh air

Prepare anti-influenza A H 1 N 1 virus HA with the method that describes in detail by embodiment 5 ₂Protein-specific chicken IgY300g makes a kind of anti-H 1 N 1 influenza sterilization sprays with fresh air of using gene engineering as main raw material.

Anti-influenza A H 1 N 1 virus HA ₂Protein-specific chicken IgY 300g

Glycerine 6,000g

PEG400 7,500g

Mentha camphor 300g

Essence 90g

Ethanol 3, and 000g (3,000ml)

Distilled water adds to 300kg (300 liters)

With anti-influenza A H 1 N 1 virus HA ₂Protein-specific chicken IgY is dissolved in the 240kg distilled water, adds the glycerine mixing.Mentha camphor, essence add dissolve with ethanol, add the PEG400 mixing again, under agitation are added on above-mentioned solution and stir evenly, filter, and add distilled water to full dose, transfer pH to 7.0, can with 0.1mol, NaOH solution.Soup is poured in the Plastic Bottle, and every bottle of 500ml examines qualified back packing entirely and promptly gets 600 bottles of anti-H 1 N 1 influenza sterilization sprayss with fresh air.

Embodiment 11: preparation anti-H 1 N 1 influenza specificity composite IgY buccal tablet

Prepare anti-influenza A H 1 N 1 virus specific IgY 1.0 grams with the method that describes in detail by embodiment 1, prepare anti-secondary infection specific IgY 1.0 grams with the method that describes in detail by embodiment 6, become anti-H 1 N 1 influenza specificity composite IgY 2.0 grams by 1: 1 mixed, with this composite IgY is main raw material, makes a kind of buccal tablet.

Anti-H 1 N 1 influenza specificity composite IgY 2.0g

Carboxymethyl cellulose 5.0g

Magnesium Stearate 0.6g

Aspartame 1.2g

Peppermint essence 0.8g

Fragrant citrus essence 1.0g

Ethanol (30% concentration) adds to the dissolving carboxymethyl cellulose

Become 1% ethanolic soln

Sorbitol Powder complements to 1,000g

Make 1,000 altogether

Concrete technical process is:

1, Sorbitol Powder and aspartame thorough mixing are even, and it is standby to cross twice of 60 mesh sieve.

2, carboxymethyl cellulose is scattered in and makes 1% ethanolic soln in 30% ethanol.

3, with 1 an amount of 2 system softwood of usefulness, 14 eye mesh screens are granulated, 60 ℃ of air seasonings, the whole grain of 18 mesh sieves.With 40 mesh sieves sift out an amount of fine powder and the abundant mixing of IgY and spray into peppermint, fragrant citrus essence stirs evenly, and admixes Magnesium Stearate again, mixes airtight every 0.6g of voltage supply sheet more than 4 hours with particle by the gross together.After the assay was approved, for packing, test entirely and dispatch from the factory.

Embodiment 12: the novel injection of preparation prevention and treatment influenza

Prepare resisiting influenza virus specific IgY 5.0g with the method that describes in detail by embodiment 1, and make the novel injection of a kind of prevention and treatment influenza.

The pure IgY 5g of resisiting influenza virus specificity

Pluronic F ₆₈ 10g

Polyvinylpyrrolidone (PVP) 10g

PGE400 110g

Polyoxyethylene Sorbitan Monooleate 10g

Water for injection adds to 1,000ml

1, water for injection adds Pluronic and PVP, heating for dissolving; Other gets Polyoxyethylene Sorbitan Monooleate, adds the PEG400 mixing; Under agitation add in the above-mentioned mixed solution; Add 0.1% needle-use activated carbon again, 60 ℃ are stirred 15min; Decarbonization filtering; 100 ℃ of 30min of the airtight heating of filtrate, it is standby to be cooled to room temperature.

2, the sterilized water for injection that activated carbon treatment crosses of learning from else's experience, in sterilising vessel, add the pure IgY lyophilized powder dissolving of resisiting influenza virus specificity, continue to stir down, be added in the above-mentioned mixed solution with thread, NaOH solution with 0.1mol is regulated pH to 6.0～7.5, supplies full dose with taking off the charcoal sterilized water for injection.

3, the sterilized 0.45 μ m 0.22 μ m millipore filtration Sterile Filtration of connecting.Embedding is in sterile chamber in aseptic filling and sealing machine.Every 2ml contains the pure IgY lyophilized powder of pure resisiting influenza virus specificity 5mg.Lamp inspection, leak detection, lettering, packing promptly get 500 IgY injections.Specification 5mg/2ml.

Embodiment 13: the IgY novel fodder additive

The anti-influenza A H 1 N 1 virus specific IgY crude extract as the fodder additives major ingredient, is made the IgY novel fodder additive.It is as follows to fill a prescription:

Raw material	Deal
Raw material	Deal	1. anti-influenza A H 1 N 1 virus specific composite IgY crude extract	350.0g
2. edible salt	10.0		350.0g
2. edible salt	10.0	3. crude dextrose	640.0g
Make	1000g	3. crude dextrose	640.0g

Concrete preparation technology is as follows:

1. it is even formula ratio anti-influenza A H 1 N 1 virus specific composite IgY crude extract and crude dextrose to be inserted in the universal powder mixing machine thorough mixing, obtains dry powder A.

2. adopt the balanced mix method that the formula ratio edible salt is added among the dry powder A, thorough mixing is even, obtains dry powder B.

3. detect qualified back packing warehouse-in.

Embodiment 14: the different H1N1virus IgY of preparation resistance hand is pressed quantitative Incapacitating sprays

The different influenza A H 1 N 1 virus specific IgY dry powder of resistance 300g adds in the pony mixer fully evenly mixed; Allocate the different influenza A H 1 N 1 virus specific IgY combination solution of 200kg (200 liters) resistance by following prescription:

The different influenza A H 1 N 1 virus specific IgY 300g of resistance

Aspartame 240g

Fragrant citrus essence 600g

Honey peach essence 200g

Peppermint essence 1400g

Distilled water adds to 200kg (200 liters)

Get distilled water 180kg (liter) earlier, add aspartame, essence then respectively, stir, slowly add IgY more while stirring; Then adding distil water stirs to 200kg (liter), measures the pH value of solution value, regulates pH to 6.5-7.5 according to the pH value of being surveyed with 1.0N NaOH solution; After preparing solution is good, must be on the pressure-pot production line manual quantitative 20000 of the Incapacitating sprays of can, after every spray tank was irritated sufficient 10ml, quantitative valve was loaded onto as boosting agent (propellent) in charged pressure nitrogen or fluorine Lyons again, sealing, promptly.

Claims

1, a kind of preparation method of anti-influenza A H 1 N 1 virus specific IgY is characterized in that, may further comprise the steps:

(S1) preparation is at the complex antigen of Influenza A H1N1;

2, method according to claim 1 is characterized in that,

Complex antigen in the described step (S1) comprises the influenza virus complex antigen, preparation process is as follows: get equivalent avian influenza vaccine liquid, porcine influenza vaccine liquid and seasonal human influenza vaccine liquid respectively, insert ultracentrifuge,, get supernatant with the 25000rpm high speed centrifugation; With conventional ammonium sulfate precipitation method precipitation virion, insert ultracentrifuge again, with the 25000rpm high speed centrifugation, get precipitation; Separate with Sepharose 4B, collect avian influenza virus, swine influenza virus and seasonal human influenza virus's particle respectively; With three kinds of influenza viruses of sucrose density gradient ultracentrifugation method purifying, specifically be to get above-mentioned three kinds of influenza virus suspension 1.0ml that slightly carry respectively, be added to gently above the sucrose liquid of 40% and 60% discontinuous density gradient, through 100, centrifugal 2 hours of 000g gets sucrose layer intermediate zone band liquid, add pH7.2,0.01M phosphate buffered saline (PBS) 1.0ml, mixing, dialysis removes sugar, promptly gets the influenza virus suspension of three kinds of purifying; By 1: 1: 1 mixed, it was even to insert the homogenizer thorough mixing again with avian influenza virus, swine influenza virus and seasonal human influenza virus's suspension of described purifying, obtained containing the compound influenza virus mixed solution of three kinds of influenza virus compositions; The compound influenza virus mixed solution of getting above-mentioned preparation respectively adds 10-15% sodium lauryl sulphate (SDS), and cracking 30 minutes obtains the compound influenza antigen composition after the cracking; Add freund adjuvant in 1: 1 ratio, insert in the high speed homogenizer, with 8, the homogenize of 000rpm high speed, form water-in-oil liquid, promptly make the influenza virus complex antigen of three kinds of influenza virus cracking compositions such as containing avian influenza virus, swine influenza virus and seasonal human influenza virus;

3, method according to claim 1 is characterized in that,

Complex antigen in the described step (S1) comprises three kinds of influenza virus polypeptide HA ₂Complex antigen, preparation process is as follows: use the RT-PCR method, respectively from H5N1 avian influenza virus, H3N2 swine influenza virus and seasonal H1N1 human influenza virus RNA clone hemagglutinin (HA ₂) remove the fragment of signal peptide and transmembrane domains in the gene; Insert the pGEM-T carrier earlier, after order-checking proved that the gene order that is obtained is correct, with restriction enzyme EcoR I and the two enzymic digestions of Not I, electrophoresis reclaimed the purpose fragment, is connected with the Yeast expression carrier pPIC9K that uses same two enzymic digestions; After transformed into escherichia coli, picking positive colony, upgrading grain, enzyme were cut and identified correctly, electricity transformed pichia yeast bacterium KM71 and GS 115; Screening positive clone on the substratum that does not contain Histidine, and then the screening height copies transformant on the substratum of the G418 that contains different concns; The single colony inoculation of picking is in substratum, and in 28 degree shaking table overnight incubation, the dilution back continues to cultivate, and treats that the light absorption value that bacterial concentration reaches OD600 is about at 0.8 o'clock, changes substratum into contain methyl alcohol substratum, continues to cultivate 24-48 hour; In the different time sampling of cultivating, with the expression amount of HA in the ELISA method mensuration supernatant, select the highest time results of expression amount, centrifugal removal cell precipitation promptly contains the great expression product in the supernatant; Through 50% ammonium sulfate precipitation, the dialysis tubing of molecular weight cut-off 10kd was dialysed 24 hours with distilled water, and behind Sephacryl S-200 and the Sephacryl S-100 column chromatography, promptly obtained three kinds of influenza virus HA of purifying respectively ₂The antigen composition; With above-mentioned three kinds of influenza virus HA ₂The antigen composition expressing protein mixed by 1: 1: 1 earlier is even, add freund adjuvant in 1: 1 ratio again, insert high speed homogenizer, with 8, the homogenize of 000rpm high speed, form water-in-oil liquid, promptly make three kinds of influenza virus HA such as containing avian influenza virus, swine influenza virus and seasonal human influenza virus ₂The complex antigen of antigen composition expressing protein.

4, method according to claim 1 is characterized in that,

Complex antigen in the described step (S1) comprises Influenza A H1N1 secondary infection bacterium complex antigen, and preparation process is as follows: with A bunch of Type B Hemolytic streptococcus (2 * 10 ⁹/ ml), streptococcus pneumoniae (2 * 10 ⁹/ ml), hemophilus influenzae (2 * 10 ⁹/ ml) and streptococcus aureus (2 * 10 ⁹/ ml) the equivalent ratio is mixed with into the malignant bacteria mixture; With the freund adjuvant of this malignant bacteria mixture in 1: 1 ratio adding equivalent, with 8,000rpm handles, and becomes water in oil mixed bacterium body fluid, makes Influenza A H1N1 secondary infection bacterium complex antigen with high speed homogenizer again;

5, according to each described method among the claim 1-4, it is characterized in that, in described step (S3), after the 4-6 by the egg yolk liquid volume extraordinarily goes into distilled water and dilutes and mix, further comprising the steps of:

Transfer pH to 5.5-6.0 with 1.0N HCI solution;

The diluent of adjusting the pH value is further stirred, it is cooled to 2-6 ℃ then, left standstill 12 hours-24 hours;

In 10, centrifugal 20 minutes of 000rpm gets separating obtained supernatant again and puts and carry out ultrafiltration and concentration 10-20 in the ultra-fine filter doubly with diluent;

Add sodium alginate solution, it is muddy to be stirred to appearance;

Add 1.0%CaCl ₂Liquid stirs, and 4 ℃ left standstill 8-12 hour;

8, centrifugal 20 minutes of 000rpm gets supernatant;

Use the 0.22 μ m membrane filtration degerming of contacting of 0.45 μ m film;

Use Ultipor VF ^TMDV50 removes virus filter and removes virus removal;

Lyophilize promptly makes anti-influenza A H 1 N 1 virus specific IgY crude extract dry powder.

6, according to each described method among the claim 1-4, it is characterized in that, in described step (S2), when bird inlay is carried out immunity, strengthen injection more once, planned immunization epidemic disease three times every two weeks; Immunity for the first time is after 20 days, and the hen of searching immunity produces immune egg, and carries out coded markings.

7, according to each described method among the claim 1-4, it is characterized in that, in described step (S4),

Get described specific IgY crude extract dry powder and be dissolved in pH7.0, the 0.01M PB liquid, successively cross ion exchange column and gel chromatography column chromatography more respectively, promptly make the pure product of specific IgY;

The bacteriophage filtering device that removes the virus filtration system that adopts U.S. Pall Ultrafine Filtration Company to make then, the thorough various bacteriophagees of filtering, the first road bacterium filtering device is to remove salmonella bacteriums such as (Salmonella) with 0.22 μ m film sterilizing filter; The second road mycoplasma filtering device is to remove the mycoplasma strainer with 0.1 μ m film to remove mycoplasma; The 3rd road virus filtering device is to use Ultipor VF ^TMDV50 removes virus filter and removes the multiple virus that comprises avian influenza virus, enterovirus, makes anti-influenza A H 1 N 1 virus specific IgY at last.

8, the preparation method of the different influenza A H 1 N 1 virus specific IgY of a kind of nanometer liposome resistance is characterized in that, may further comprise the steps:

Get the dry powder of the different influenza A H 1 N 1 virus specific IgY of resistance that makes according to claim 2 or 3 described methods, add pulverizings of milling in the supper micron mill, be processed into size between 1-100nm, granularity is above 〉=15000 purpose ultramicrons;

9, the different influenza A H 1 N 1 virus specific composite IgY of a kind of resistance is characterized in that its composition comprises:

(1) according to the different influenza A H 1 N 1 virus specific IgY of resistance of claim 2 or 3 described method preparations or the different influenza A H 1 N 1 virus specific IgY of nanometer liposome resistance of described according to Claim 8 method preparation,

(2) the anti influenza secondary infection bacterium specific IgY for preparing according to the described method of claim 4,

Above-mentioned two kinds of compositions are pressed (1～10): the mixed of (1～10) is even, promptly obtains the different influenza A H 1 N 1 virus specific composite IgY of resistance.

10, a kind of preparation at Influenza A H1N1 is characterized in that, comprising in following four kinds of compositions one or more:

(1) according to the different influenza A H 1 N 1 virus specific IgY of resistance of claim 2 or the preparation of 3 described methods;

(2) the anti influenza secondary infection bacterium specific IgY for preparing according to the described method of claim 4;

(3) the different influenza A H 1 N 1 virus specific IgY of nanometer liposome resistance of described according to Claim 8 method preparation;

(4) the different influenza A H 1 N 1 virus specific composite IgY of the described resistance of claim 9.

11, the various preparations at Influenza A H1N1 according to claim 10 is characterized in that, also comprise the compound composition that pharmaceutical chemicals and/or Chinese medicine are made into;

Above-mentioned composition is equipped with distilled water, be deployed into the solvent of specific IgY concentration 0.01-20.0%, the human preparation at Influenza A H1N1 that utilizes described solvent to make comprises: inhaling type propellant, nasal spray, mouth spraying agent, nasal drop, eye drops, throat spraying agent, injection and air sanitizer;

Perhaps, above-mentioned composition is equipped with various auxiliary materials, and the human preparation of making at Influenza A H1N1 comprises: buccal tablet, chewable tablet, capsule, pulvis;

Perhaps, above-mentioned composition is equipped with distilled water, be deployed into the solvent of specific IgY concentration 0.01-20.0%, utilize described solvent to make the preparation that uses for Mammals such as pig, ox, sheep and bird and comprise: nasal spray, mouth spraying agent, nasal drop, eye drops, injection and air sanitizer, liquid feed additive at Influenza A H1N1;

Perhaps, above-mentioned composition is equipped with various auxiliary materials, makes the comprising at Influenza A H1N1 of using for Mammals such as pig, ox, sheep and bird: pulvis, fodder additives.