CN104826093A - Th2 immune response antagonist for treating chronic infectious hepatopathy, and yolk antibody thereof - Google Patents

Abstract

The invention discloses a Th2 immune response antagonist for treating chronic infectious hepatopathy. The antagonist adopts one or more of an antibody resisting Th2 type cytokines and acceptors thereof, a vaccine prepared by using the antigen components of the Th2 type cytokines and/or the acceptors thereof, a soluble acceptor, a cytokine mutant losing physiologic activity and still having activity combined with the Th2 type cytokine acceptors, a polypeptide or oligopeptide derived from the Th2 type cytokine or the acceptor thereof, or an antibody resisting cells generating the Th2 type cytokines as an effective component. The invention also discloses a yolk antibody for preventing or/and treating the chronic infectious hepatopathy. The chronic infectious hepatopathy is treated by inhibiting Th2 immune response for the first time. The Th2 immune antagonist provided by the invention has the advantages of substantial curative effect, safety and no toxic side effects.

Description

Be used for the treatment of Th2 immunoreation antagonist and the yolk antibody of chronic infectious hepatopathy

Technical field

The present invention relates to medicine and biological technical field, more specifically, the present invention relates to a kind of the Th2 immunoreation antagonist and the yolk antibody that are used for the treatment of infectious liver disease.

Background technology

Chronic infectious hepatitis refers to the liver chronic inflammation disease that the course of disease is caused by pathogenic microorganism more than half a year, the pathogenesis of various chronic infectious hepatopathy is different, mainly can be divided into chronic hepatitis B (CHB), chronic hepatitis C (CHC) and chronic hepatitis E etc.Chronic hepatitis can cause liver cirrhosis, liver failure and hepatocarcinoma, has a strong impact on the healthy of people.

Chronic infectious incidence of hepatitis mechanism is complicated, closely related with the immune state of body, CD4+ help property T cell (i.e. Th cell) is the important adjustment cell of body, difference according to its cytokine produced mainly can be divided into the hypotypes such as Th1 and Th2, participates in respectively regulating cellular immunization and humoral immunization.By effectively suppressing Th2 immunoreation can as of a chronic infectious treating hepatitis new thinking.

Interleukin-4 (IL-4) is that induction produces the immunoreactive major cytokine of Th2, if can reduce the content of IL-4 in host, can effectively suppress Th2 immunoreation, thus promote the immunoreactive generation of Th1.Experiment proves, injects anti-IL-4 antibody or use IL-4 vaccine can reach this object to host.Because IL-4 only has with its receptor (R) in conjunction with competence exertion biological effect in humans and animals body, any antagonist that can block IL-4 and its receptors bind all can effectively suppress Th2 immunoreation, at present, certified antagonist has the IL-4 mutant losing physiologically active, the polypeptide coming from IL-4, solubility IL-4R α (sIL-4R α), anti-IL-4R antibody, IL-4 and IL-4R vaccine etc.Because IL-4 produces primarily of CD4+T cell, use anti-CD4+T cell antibody also effectively can suppress the generation of IL-4.

Interleukin-13 (IL-13) is the cytokine with IL-4 with similar functions, primarily of CD4+ helper T lymphocyte (Th2 cell) secretion activated.IL-l3 receptor has two types: IL-13 receptor alpha-1 (IL-13R α 1) and IL-13 receptor alpha-2 (IL-13R α 2).In IL-13R, IL-13R α 1 and IL-4R α exists with homodimer form, and IL-4R α makes IL-13R α 1 significantly improve with the affinity of IL-13.Just because of IL-13 and IL-4 has the characteristic of shared same signal of interest transduction functional receptor component, both biological functions are made to have certain similarity.Suppress the immunoreactive method of Th2 to can be used for IL-13 and IL-13 receptor equally above by signal transduction links such as adjustment IL-4 and IL-4 receptors, reach the similar immunoreactive object of suppression Th2.Principle according to this, the epitaxy technology that can produce similar effect also can expand other slightly secondary Th2 type cytokines further to, as IL-5 and IL-5 receptor, IL-6 and IL-6 receptor, IL-10 and IL-10 receptor etc., and on shift onto by suppressing the cell producing Th2 type cytokines to realize, as suppressed CD4+T cytoactive etc.

But the method at present by suppressing Th2 immunoreation to carry out disease therapy, is rarely seenly applied to asthma, there is not been reported for the application in treatment chronic infectious hepatitis etc.

Yolk immunoglobulin (Immunoglobulin of egg yolk, IgY), also known as yolk antibody, that immunoglobulin in hen blood is passed to Ovum Gallus domesticus Flavus and the immunoglobulin shielded to chick after chick is hatched in process and hatched, the structure of IgY is similar to IgG, there is typical IgG space conformation, can be combined with specific antigen, a lot of aspect is better than mammiferous IgG, the specific antibody IgY obtained by antigen immune bird inlay can be used for preparing biological diagnosis reagent and treatment preparation etc., and IgY has the following advantages: hen is easy to raise, collect egg instant, without the need to taking out animal blood, not damaged, meet modem animal protection philosophy, and productive rate is high, cost is low, good stability, high specificity, is therefore with a wide range of applications in immunologic diagnosis, disease prevention etc.But have not yet to see about the report of yolk immune protein for prevention and therapy human or animal chronic infectious hepatopathy.

Summary of the invention

For above-mentioned prior art, the object of this invention is to provide a kind of Th2 immunoreation antagonist that can be used for treating chronic infectious hepatopathy.

Another object of the present invention is to provide and a kind ofly can be used for preventing or/and the high immunity yolk antibody for the treatment of chronic infectious hepatopathy.

For achieving the above object, the present invention adopts following technical scheme:

A kind of Th2 immunoreation antagonist that can be used for treatment chronic infectious hepatopathy, vaccine, the soluble recepter made with the antigenic component (comprising gene) of the antibody of anti-Th2 type cytokines and receptor thereof, Th2 type cytokines and/or its receptor, lose physiologically active but still have and the cytokine mutant of Th2 type cytokines receptor-binding activity, the polypeptide coming from Th2 type cytokines or its receptor or oligopeptide, or one in the antibody of the cell of anti-generation Th2 type cytokines or several be arbitrarily effective ingredient.

Described Th2 type cytokines is IL-4, IL-13, IL-5, IL-6 or IL-10;

The receptor of described Th2 type cytokines is IL-4R α, alpha-1 (IL-13R α 1), alpha-2 (IL-13R α 2), IL-5 receptor, IL-6 receptor or IL-10 receptor;

Describedly lose physiologically active but still there is the cytokine mutant with Th2 type cytokines receptor-binding activity, refer to by exist gene order lack mRNA coded by protein, as the aminoacid after Mus IL-4 one of carbon tip 118 is clipped completely the mutant IL-4T118 that produces namely lose IL-4 activity, but still there is the activity of its corresponding receptors bind;

Described polypeptide or the oligopeptide coming from Th2 type cytokines or its receptor, there is the activity be combined with corresponding cytokine receptor, or there is the activity combined with corresponding cytokine of vying each other to receptor, thus reduce the physiologically active of Th2 type cytokines;

The cell of described generation Th2 type cytokines is CD4+T cell;

The vaccine that the antigenic component (comprising gene) of described Th2 type cytokines and/or its receptor is made does with the natural of Th2 type cytokines or the intact proteins of synthetic or the protein fragments containing epitope any type of protein vaccine or coupling protein vaccine that antigen makes;

Or: do with the genetic fragment of the complete genome of Th2 type cytokines or antigen expressed epi-position any type of gene vaccine or fusion gene vaccine that antigen gene or major antigen gene make.

The present invention still further provides and a kind ofly can be used for preventing or/and the high immunity yolk antibody for the treatment of chronic infectious hepatopathy, with Th2 immunoreation antagonist for effective ingredient makes vaccine, immunity is carried out to hen, the egg that after collecting immunity, hen produces, makes ELISA and tires higher than the high immunity yolk antibody of 1:10000.

The preparation method of this high immunity yolk antibody, step is as follows:

With Th2 immunoreation antagonist for effective ingredient makes vaccine, carry out immunity to hen, immune programme for children is: it is 1 part/only that head exempts from dosage; Carry out two after head exempts from 3 weeks to exempt from, dosage is 2 parts/only; Head exempts from 6 Zhou Housan and exempts from, and dosage is 2 parts/only; In every part vaccine, the content of Th2 immunoreation antagonist is 50-100 μ g; Two exempt to exempt from three to measure for latter 20 days the yolk antibody that immune hen lays eggs tires, ELISA tire be judged to be higher than 1:10000 qualified; Otherwise proceed the 4th immunity, immunizing dose is 4 parts/only, four exempt to measure for latter 20 days the yolk antibody that immune hen lays eggs tires; Collect ELISA to tire higher than the egg of 1:10000, make high-immunity yolk egg albumen powder, to obtain final product.

The preparation of described high-immunity yolk egg albumen powder, specifically comprises the following steps:

(1) shell of being tired by the ELISA of collection higher than the egg of 1:10000 carries out disinfection, collection egg yolk liquid of beating eggs; Sterilization adopts volume fraction to be the ethanol of 70%;

(2) egg yolk liquid of step (1) is carried out frozen drying or high temperature instantaneous pressure spraying dry, make high-immunity yolk egg albumen powder.

In step (2), the spray-dired temperature of high temperature instantaneous pressure is 160 DEG C.

Beneficial effect:

(1) system of the present invention first passage suppresses Th2 immunoreation to treat chronic infectious hepatitis, and provide one group of Th2 immunity antagonist, it is evident in efficacy, safe without toxic side effect.

(2) present invention also offers a kind of high-immunity yolk egg albumen powder for prevention and therapy chronic infectious hepatitis, can be used as a kind of health promoting product to eat, safety is high, nutritious, and test proves, this high-immunity yolk egg albumen powder significantly can reduce the viral infection amount of hepatitis C patient.

Detailed description of the invention

Below in conjunction with embodiment, the present invention is further illustrated.

Embodiment 1: prepared by structure and the vaccine of mice IL-4, IL-4R α and IL-13 genetic vaccine

According to full genome or the portion gene coded sequence of mice IL-4, IL-4R α and IL-13, design a pair suitable primer respectively, to be increased respectively IL-4, IL-4S (comprising one section of polypeptide be made up of 14 aminoacid of IL-4 epitope), all or part of cDNA sequence of IL-4R α and IL-13 by RT-PCR method.Then, through determined dna sequence, after the cDNA sequence that proof increases is accurate, again IL-4, IL-4R α and IL-4S, IL-13cDNA are cloned into respectively the suitable position of pET-30 and pET-32 expression vector, and further recombiant plasmid and pET-30 or pET-32 expression vector blank plasmid are proceeded in suitable expression coli strain, through scale fermentation, extraction purification mice IL-4, IL-4R α albumen and IL-4S, IL-13 fusion rotein and vehicle Control albumen.It is [different according to the content of purification IL-4, IL-4R α albumen and IL-4S, IL-13 fusion rotein after purified mouse IL-4, IL-4R α albumen is mixed by proper proportion with oily adjuvant or aluminium glue adjuvant etc. respectively from the albumen expressed by IL-4S, IL-13 fusion rotein or vehicle Control, for oil-adjuvant vaccine, the volume ratio of oil/protein solution can be controlled at (1:1) ~ (3:1); For aluminium glue Adjuvanted vaccines, the volume ratio of aluminium glue/protein solution can be controlled at (1:1) ~ (1:5); Require that the amount of IL-4, IL-4R α albumen or IL-4S, IL-13 fusion rotein or vehicle Control albumen in every part vaccine is about 15-20 μ g; The volume of every part vaccine is about 0.1ml; Or according to the preparation of veterinary's pharmacopeia rule of operation], experiment vaccine is made in further homogenate emulsifying (being undertaken by the rule of operation of refiner used).

Described oily adjuvant is by the pale yellow oily liquid body formed after the Span-80 of 94% (V/V) mineral oil (No. 10 white oils), 6% (V/V), 2% (g/V) aluminium stearate heating for dissolving; Oil adjuvant is the milky water-in-oil type vaccine of the thickness that vaccine protein solution and oily adjuvant are formed through emulsifying.General employing injection inoculation, being product conventional in prior art, is in animal inactivated vaccine, use wider adjuvant at present.

Described aluminium glue adjuvant also uses wider adjuvant in animal inactivated vaccine.

The construction and expression of embodiment 2: mouse soluble IL-4R α (sIL-4R α) and truncated-type IL-4 (IL-4-T118S) fusion expression plasmid

According to full genome or the portion gene coded sequence of mice IL-4R α and IL-4, design a pair suitable primer, front 118 aminoacid cDNA sequence of increased respectively by RT-PCR method main IL-4R α cDNA sequence (50 aminoacid) and IL-4.Then, through determined dna sequence, after the cDNA sequence that proof increases is accurate, be cloned into the suitable position of pET-32 fusion expression vector again, and further recombiant plasmid and pET-32 expression vector blank plasmid are proceeded in suitable expression coli strain, through scale fermentation, extraction purification mice sIL-4R α and IL-4-T118S fusion rotein and vehicle Control albumen.

Embodiment 3: safety testing and the anaphylaxis of mice IL-4, IL-4S, IL-4R α and IL-13 genetic vaccine, mice sIL-4R α and IL-4-T118S fusion rotein and vehicle Control albumen are tested

By the method for embodiment 1, be mixed with into IL-4, IL-4R α albumen or IL-4S, IL-13 amalgamation protein vaccine or vehicle Control protein vaccine with oily adjuvant or aluminium glue adjuvant by the volume ratio of 1:1 by after the albumen normal saline (or PBS) expressed by purification IL-4, IL-4R α albumen or IL-4S, IL-13 fusion rotein or vehicle Control suitably dilution.IL-4, IL-4R α albumen prepared or IL-4S, IL-13 amalgamation protein vaccine divide low (0.1ml, about 1 part), in (0.25ml, about 2.5 parts), high (0.5ml, about 5 parts) three various dose groups immune BALB/c mouse respectively, separately establish carrier protein vaccine and PBS immunized controls group, often organize 5 BALB/c mouse, subcutaneous multi-point injection.3 weeks, interval, carries out second time and third time inoculation respectively.Observe after vaccine injection that mice has no adverse reaction, the phenomenon such as irritated, dead respectively.Result shows: physically well develop after mouse inoculation vaccine, and the mental status and appetite are all normal, and inoculation position has the untoward reaction such as transient swelling, heating, especially more obvious in high dose group performance.For oily adjuvant Seedling, high dose group mice, because vaccine absorbs comparatively slow, has slight granuloma to react.

Similar with the experimental technique of above-mentioned vaccine, the mouse soluble IL-4R α (sIL-4R α) of purification and IL-4-T118S fusion rotein and vehicle Control albumen are divided low (0.5 μ g), in (5 μ g), high (50 μ g) three various dose groups, separately establish saline control group, often organize 5 BALB/c mouse, in hind leg muscle or tail vein injection.3 weeks, interval, carries out second time and third time injection respectively.Observe after injection that mice has no adverse reaction, the phenomenon such as irritated, dead respectively.Result shows: physically well develop after injected in mice albumen, and the mental status and appetite are all normal, do not find obvious adverse reaction.

Embodiment 4: mice IL-4, IL-4S, IL-4R α and IL-13 genetic vaccine, mouse soluble IL-4R α (sIL-4R α) and IL-4-T118S fusion rotein strengthen the test that mice prevents and treats chronic hepatitis B

By the method for embodiment 1, after the albumen normal saline expressed by IL-4, IL-4R α albumen of purification or IL-4S, IL-13 fusion rotein or vehicle Control is suitably diluted and aluminium glue adjuvant be mixed with in the ratio of 1:1 and test or control vaccine.Require that the amount of IL-4, IL-4R α albumen or IL-4S, IL-13 fusion rotein or vehicle Control albumen in every part vaccine is about 15-20 μ g; The volume of every part vaccine is about 0.1ml.To experiment mice respectively at first 3 weeks of hepatitis B virus inoculation and 1 week abdominal cavity (i.p.) inoculate IL-4, IL-4R α albumen prepared by embodiment 1 and IL-4S, IL-13 antigen-4 fusion protein gene and to recombinate aluminium glue vaccine, separately establish carrier protein aluminium glue vaccine and PBS immunized controls group.To every mouse tail vein inoculation Hepatitis B virus-DNA equivalent (viral genome equivalents) 5 × 10 ¹⁰.Inoculate latter 14 days tail vein bloods, ELISA method measures the content of hepatitis B surface antigen (HbsAg).Result shows, IL-4, IL-4R α albumen and IL-4S, IL-13 antigen-4 fusion protein gene recombiant vaccine all significantly can reduce the infection (see table 1) of hepatitis B virus.

For mouse soluble IL-4R α (sIL-4R α) and IL-4-T118S fusion rotein treatment group, to experiment mice respectively at after inoculation hepatitis B virus at interval of 4 days in mouse tail vein injection 5 μ g sIL-4R α or IL-4-T118S fusion rotein or fusion rotein contrast.Inoculate latter 14 days tail vein bloods, ELISA method measures the content of hepatitis B surface antigen (HbsAg).Result shows, sIL-4R α and IL-4-T118S fusion rotein treat the infection (see table 1) that significantly can reduce hepatitis B virus.Table 1: mice (C57BL/6, ♀) IL-4, IL-4R α albumen and IL-4S, IL-13 antigen-4 fusion protein gene recombiant vaccine group, mice sIL-4R α and IL-4-T118S fusion rotein treatment group all can strengthen the result of the test that mice anti-hepatitis virus infects and (often organize 10, data are HbsAg content meansigma methods, unit: ng/ml)

Embodiment 5: the preparation of anti-mouse and people IL-4, IL-4S, IL-4R α and IL-13 chicken high immunity yolk antibody (egg albumen powder)

By the method for embodiment 1, after respectively the albumen normal saline expressed by the mice of purification and people IL-4, IL-4R α albumen or IL-4S, IL-13 fusion rotein or vehicle Control suitably being diluted and aluminium glue adjuvant be mixed with in the ratio of 1:1 and test or control vaccine, require that the amount of IL-4, IL-4R α albumen or IL-4S, IL-13 fusion rotein or vehicle Control albumen in every part vaccine is about 50-100 μ g, the volume of every part vaccine is about 0.5ml.Immune programme for children is as follows: select the healthy hens just having opened product to be laboratory animal, random packet, often organize 50.It is 1 part/only that head exempts from dosage; Head to exempt from after 3 weeks two and exempts from, and dosage is 2 parts/only; Head exempts from 6 Zhou Housan and exempts from, and dosage is 3 parts/only; Two exempt to exempt from three latter 20 days respectively group random collecting 10 pieces of eggs respectively, measure tiring of yolk antibody, if ELISA tire be judged to higher than 1:10000 qualified; Otherwise, proceed the 4th immunity, immunizing dose to be dosage be 4 parts/only; Four exempt from latter 20 days respectively group random collecting 10 pieces of eggs respectively, measure tiring of yolk antibody, if ELISA tire be judged to higher than 1:10000 qualified.Collect qualified egg, prepare high-immunity yolk egg albumen powder with frozen drying or instantaneous high-temperature spray drying method, be placed on cold drying place, for subsequent use.

Embodiment 6: anti-mouse IL-4, IL-4S, IL-4R α and IL-13 chicken high immunity yolk antibody (egg albumen powder) strengthen the test that mice prevents and treats chronic hepatitis B

Within front 1 week, to start to drink water high immunity yolk antibody prepared by Application Example 5 respectively at hepatitis B virus inoculation to experiment mice, by 1% dilution, freely drink water, to testing and terminating.To every mouse tail vein inoculation Hepatitis B virus-DNA equivalent (viralgenome equivalents) 5 × 10 ¹⁰.Inoculate latter 14 days tail vein bloods, ELISA method measures the content of hepatitis B surface antigen (HbsAg).Result shows: anti-IL-4, IL-4S, IL-4R α and IL-13 chicken high immunity yolk antibody (egg albumen powder) all significantly can reduce the infection (see table 2) of hepatitis B virus.

Table 2: anti-IL-4, IL-4S, IL-4R α and IL-13 chicken high immunity yolk antibody (egg albumen powder) strengthens result of the test (the female Mus of C57BL/6 of mice control chronic hepatitis B, often organize 10, data are HbsAg content meansigma methods, unit: ng/ml)

Embodiment 7: anti-human IL-4, IL-4S, IL-4R α and IL-13 chicken high immunity yolk antibody (egg albumen powder) strengthens the test of hepatitis C Rehabilitation

Give people's high immunity yolk antibody prepared by the oral embodiment 5 of the cold boiled water of hepatitis C patient respectively, by every day 1 time, the amount of each 5 grams is taken, and continues application 30 days.With latter three weeks of experiment before experiment, gather the serum of patient respectively, measure the content of each group of hepatitis C virus with quantitative RT-PCR respectively.Result shows: anti-IL-4, IL-4S, IL-4R α and IL-13 chicken high immunity yolk antibody (egg albumen powder) all significantly can reduce the viral infection amount (see table 3) of hepatitis C patient.

Table 3: anti-human IL-4, IL-4S, IL-4R α and IL-13 chicken high immunity yolk antibody (egg albumen powder) strengthens the result of the test (quantitative RT-PCR, data are HCV rna content, unit: copies/ml) of hepatitis C Rehabilitation

Claims (10)

1. one kind is used for the treatment of the Th2 immunoreation antagonist of chronic infectious hepatopathy, it is characterized in that, vaccine, the soluble recepter made with the antigenic component of the antibody of anti-Th2 type cytokines and receptor thereof, Th2 type cytokines and/or its receptor, lose physiologically active but still have and the cytokine mutant of Th2 type cytokines receptor-binding activity, the polypeptide coming from Th2 type cytokines or its receptor or oligopeptide, or one in the antibody of the cell of anti-generation Th2 type cytokines or several be arbitrarily effective ingredient.

2. the Th2 immunoreation antagonist being used for the treatment of chronic infectious hepatopathy according to claim 1, it is characterized in that, described Th2 type cytokines is IL-4, IL-13, IL-5, IL-6 or IL-10.

3. the Th2 immunoreation antagonist being used for the treatment of chronic infectious hepatopathy according to claim 1, is characterized in that, the receptor of described Th2 type cytokines is IL-4R α, alpha-1, alpha-2, IL-5 receptor, IL-6 receptor or IL-10 receptor.

4. the Th2 immunoreation antagonist being used for the treatment of chronic infectious hepatopathy according to claim 1, it is characterized in that, describedly lose physiologically active but still there is the cytokine mutant with Th2 type cytokines receptor-binding activity, referring to the protein coded by the aminoacid that there is sequence deletion.

5. the Th2 immunoreation antagonist being used for the treatment of chronic infectious hepatopathy according to claim 1, it is characterized in that, described polypeptide or the oligopeptide coming from Th2 type cytokines or its receptor, there is the activity be combined with corresponding cytokine receptor, or there is the activity combined with corresponding cytokine of vying each other to receptor, thus reduce the physiologically active of Th2 type cytokines.

6. the Th2 immunoreation antagonist being used for the treatment of chronic infectious hepatopathy according to claim 1, is characterized in that, the cell of described generation Th2 type cytokines is CD4+T cell.

7. the Th2 immunoreation antagonist being used for the treatment of chronic infectious hepatopathy according to claim 1, it is characterized in that, the vaccine that the antigenic component of described Th2 type cytokines and/or its receptor is made does with the natural of Th2 type cytokines or the intact proteins of synthetic or the protein fragments containing epitope any type of protein vaccine or coupling protein vaccine that antigen makes;

Or: do with the genetic fragment of the complete genome of Th2 type cytokines or antigen expressed epi-position any type of gene vaccine or fusion gene vaccine that antigen gene or major antigen gene make.

8. one kind for preventing or/and treat the yolk antibody of chronic infectious hepatopathy, it is characterized in that, with the Th2 immunoreation antagonist of claim 1 for effective ingredient makes vaccine, immunity is carried out to hen, the egg that after collecting immunity, hen produces, makes ELISA and tires higher than the high immunity yolk antibody of 1:10000.

9. according to claim 8 for preventing or/and treat the preparation method of the yolk antibody of chronic infectious hepatopathy, it is characterized in that, step is as follows:

With Th2 immunoreation antagonist for effective ingredient makes vaccine, carry out immunity to hen, immune programme for children is: it is 1 part/only that head exempts from dosage; Carry out two after head exempts from 3 weeks to exempt from, dosage is 2 parts/only; Head exempts from 6 Zhou Housan and exempts from, and dosage is 2 parts/only; In every part vaccine, the content of Th2 immunoreation antagonist is 50-100 μ g; Two exempt to exempt from three to measure for latter 20 days the yolk antibody that immune hen lays eggs tires, ELISA tire be judged to be higher than 1:10000 qualified; Otherwise proceed the 4th immunity, immunizing dose is 4 parts/only, four exempt to measure for latter 20 days the yolk antibody that immune hen lays eggs tires; Collect ELISA to tire higher than the egg of 1:10000, make high-immunity yolk egg albumen powder, to obtain final product.

10. preparation method according to claim 9, is characterized in that, the preparation of described high-immunity yolk egg albumen powder, specifically comprises the following steps:

(1) shell of being tired by the ELISA of collection higher than the egg of 1:10000 carries out disinfection, collection egg yolk liquid of beating eggs; Sterilization adopts volume fraction to be the ethanol of 70%;

(2) by the egg yolk liquid frozen drying of step (1) or instantaneous high-temperature spraying dry, high-immunity yolk egg albumen powder is made.