阅读说明：本技术 一种鸭病毒性肝炎卵黄抗体及其制备方法 (Duck viral hepatitis egg yolk antibody and preparation method thereof ) 是由 刘国英 陈生雷 吴冠辰 李慧 姜斌 杨淑芳 佟晖 杨阔 舒秀伟 于 2020-11-18 设计创作，主要内容包括：本发明公开了一种用于治疗、预防、减缓或控制鸭病毒性肝炎的卵黄抗体的制备方法,包括如下步骤：将微生物保藏号为CGMCC No.19696的Ⅰ型鸭病毒性肝炎病毒疫苗株和/或微生物保藏号为CGMCC No.19695的Ⅲ型鸭病毒性肝炎病毒疫苗株作为免疫原,制备成鸭病毒性肝炎疫苗；用鸭病毒性肝炎疫苗接种禽体,获得高免禽蛋；从高免禽蛋中提取卵黄抗体。本发明还公开了通过前述方法制备得到的卵黄抗体。该卵黄抗体特异性好,安全有效。(The invention discloses a preparation method of a yolk antibody for treating, preventing, slowing down or controlling duck viral hepatitis, which comprises the following steps: preparing duck viral hepatitis vaccine by using I type duck viral hepatitis virus vaccine strain with microorganism preservation number of CGMCC No.19696 and/or III type duck viral hepatitis virus vaccine strain with microorganism preservation number of CGMCC No.19695 as immunogen; inoculating the poultry with a duck viral hepatitis vaccine to obtain a high-immunity poultry egg; yolk antibodies are extracted from the high-immunity poultry eggs. The invention also discloses the yolk antibody prepared by the method. The yolk antibody has good specificity, and is safe and effective.)

1. A preparation method of a yolk antibody for treating, preventing, slowing down or controlling duck viral hepatitis comprises the following steps: immunizing an avian body by taking a duck viral hepatitis virus vaccine strain as an immunogen to prepare the egg yolk antibody;

the duck viral hepatitis virus vaccine strain is a duck viral hepatitis virus I vaccine strain and/or a duck viral hepatitis virus III vaccine strain;

the I-type duck viral hepatitis virus vaccine strain is a virus strain with the microbial preservation number of CGMCC No.19696, or a passage virus strain or a mutant virus strain with unchanged clinical pathogenicity and immunogenicity;

the III type duck viral hepatitis virus vaccine strain is a virus strain with the microorganism preservation number of CGMCC No.19695, or a passage virus strain or a mutant virus strain with unchanged clinical pathogenicity and immunogenicity.

2. The method of claim 1, comprising the steps of:

(a) preparation of the vaccine:

taking the duck viral hepatitis virus vaccine strain I and/or the duck viral hepatitis virus vaccine strain III as an immunogen to prepare a duck viral hepatitis virus vaccine;

(b) preparing high-immunity poultry eggs:

inoculating the duck viral hepatitis virus vaccine to the poultry body to obtain a high-immunity poultry egg;

(c) extraction of yolk antibody:

and extracting yolk antibodies from the hyperimmune eggs.

3. The method of claim 2, wherein in step (a), the duck viral hepatitis virus vaccine comprises the immunogen and a vaccine adjuvant;

preferably, the duck viral hepatitis virus vaccine is an inactivated vaccine;

preferably, in step (a), the vaccine adjuvant comprises a lipid adjuvant, a first emulsifier and a second emulsifier;

preferably, in step (a), the lipid adjuvant is selected from white oil;

preferably, in step (a), the first emulsifier is selected from span-80;

preferably, in step (a), the second emulsifier is selected from tween-80;

preferably, in the step (a), the components of the duck viral hepatitis virus vaccine comprise the following components in the ratio of dosage:

the type I duck viral hepatitis virus vaccine strain: type III duck viral hepatitis virus vaccine strain: oil and fat adjuvants: first emulsifier: 750-^7.0ELD₅₀:750-2250×10^7.0ELD₅₀:150-400g:10-30g:5-20g；

Preferably, in the step (a), the components of the duck viral hepatitis virus vaccine comprise the following components in the ratio of dosage:

the type I duck viral hepatitis virus vaccine strain: oil and fat adjuvants: first emulsifier: 4500 × 10 ═ 1500-^7.0ELD₅₀:150-400g:10-30g:5-20g；

Preferably, in the step (a), the components of the duck viral hepatitis virus vaccine comprise the following components in the ratio of dosage:

type III duck viral hepatitis virus vaccine strain: oil and fat adjuvants: first emulsifier: 4500 × 10 ═ 1500-^7.0ELD₅₀:150-400g:10-30g:5-20g；

Preferably, in step (a), the ELD of said duck viral hepatitis virus type I vaccine strain and/or said duck viral hepatitis virus type III vaccine strain₅₀Is calculated according to a Reed-Muench method based on duck embryo culture;

preferably, in the step (a), the preparation of the duck viral hepatitis virus vaccine comprises the following steps:

(a1) preparation of oil phase adjuvant:

mixing the oil-fat adjuvant with a first emulsifier to obtain an oil-phase adjuvant;

(a2) preparation of the aqueous phase:

mixing the inactivated duck viral hepatitis virus vaccine strain with a second emulsifier to obtain a water phase;

(a3) mixing:

mixing the oil phase adjuvant with the water phase to obtain the vaccine composition.

4. The method of claim 2, wherein in step (b), the high-immunity poultry egg is prepared by the steps of:

(b1) basic inoculation:

inoculating the duck viral hepatitis virus vaccine to poultry in an egg laying period;

(b2) and (3) strengthening inoculation:

after 10-28 days of basic inoculation, the duck viral hepatitis virus vaccine is used for inoculating poultry animals in the laying period;

(b3) enhanced inoculation:

after 10-28 days of intensive inoculation, the duck viral hepatitis virus vaccine is used for inoculating poultry animals in the laying period to obtain the hyperimmune eggs;

preferably, the avian animal is selected from: chicken, duck, goose;

preferably, the egg-laying period is 120-400 days old;

preferably, the basal inoculation is subcutaneous injection containing 1.5-6.0X 10^7.0ELD₅₀Said duck viral hepatitis virus vaccine of virus;

preferably, the booster inoculation is intramuscular injection containing 3.0-12.0X 10^7.0ELD₅₀Said duck viral hepatitis virus vaccine of virus;

preferably, the booster inoculation intramuscular injection contains 3.0 to 12.0X 10^7.0ELD₅₀Said duck viral hepatitis virus vaccine of virus;

preferably, in step (b), the neutralizing antibody titer against the duck viral hepatitis virus type I in the hyperimmunized egg, as determined by an egg neutralization test, is greater than or equal to 1:2048 and/or the neutralizing antibody titer against the duck viral hepatitis type III virus is more than or equal to 1:2048, mixing the two solutions;

preferably, in the step (c), the yolk antibody is extracted by the following steps:

(c1) separating egg yolk:

obtaining yolk of the high-immunity poultry egg;

(c2) and (3) extraction:

extracting the yolk to obtain the crude yolk antibody;

preferably, prior to step (c1), the egg yolk is separated after sterilizing the hyperimmunized egg;

preferably, the method for disinfecting the high-immunity poultry eggs is as follows:

disinfecting the high-immunity poultry eggs by using a benzalkonium bromide aqueous solution;

preferably, the concentration of the aqueous benzalkonium bromide solution is 0.08-0.12 wt%;

preferably, disinfecting the high-immunity poultry eggs by using a new benzalkonium chloride aqueous solution at the temperature of 35-45 ℃;

preferably, the method of disinfecting poultry eggs further comprises:

hermetically fumigating the hyperimmune poultry disinfected by the benzalkonium bromide aqueous solution with formalin;

preferably, the time of fumigation is 20-40 minutes;

preferably, in step (c1), manually or instrumenting the hyperimmunized egg, removing egg white, blastoderm and frenulum, collecting the yolk;

preferably, in step (c2), the step of extracting is:

mixing the yolk with acidified water to obtain a yolk mixed solution;

centrifuging the yolk mixed solution, and collecting supernatant to obtain the crude yolk antibody;

preferably, in step (c2), the acidified water has a pH of 3.5 to 4.5;

preferably, in step (c2), the volume ratio of the egg yolk to the acidified water is 1: 4-8;

preferably, in step (c2), the mixing is at rest for 3-8 hours at 2-5 ℃;

preferably, in step (c2), the mixing is carried out under stirring conditions;

preferably, in step (c2), the rotation number of the centrifugation is 6000-10000 rpm;

preferably, in step (c2), the time of centrifugation is 10-40 minutes;

preferably, in the step (c), the yolk antibody extraction step further comprises:

(c3) treating the crude yolk antibody with a clarifying agent to obtain clarified yolk antibody;

preferably, the clarifying agent is n-octanoic acid;

preferably, in step (c3), the treatment time with the clarifying agent is 5 to 10 hours;

preferably, in step (c3), the amount of the clarifying agent in the crude egg yolk antibody is 0.05-0.1 v/v%;

preferably, in the step (c), the yolk antibody extraction step further comprises:

(c4) purification of yolk antibody:

purifying the clarified yolk antibody to obtain the purified yolk antibody;

preferably, in the step (c4), the yolk antibody is purified by the steps of:

filtering and clarifying the clarified egg yolk antibody;

preferably, in step (c4), the clarifying step is filtering with a 0.5 μm filter element to obtain a filtrate;

preferably, in the step (c4), the yolk antibody purification step further comprises:

filtering and sterilizing the filtrate obtained by filtering and clarifying;

preferably, in step (c4), the filter sterilization is performed by filtering with a 0.22 μm filter element to obtain a filtrate;

preferably, in the step (c), the yolk antibody extraction step further comprises: (c5) concentration:

concentrating the egg yolk antibody solution to obtain an egg yolk antibody concentrated solution;

preferably, the concentration is obtained after the ultrafiltration membrane with the molecular weight cut-off of 80-120KD is concentrated;

preferably, in the yolk antibody concentrated solution, the neutralizing antibody titer against the duck viral hepatitis virus type I is determined by a chicken embryo neutralization test method and is more than or equal to 1:8192, and/or the neutralizing antibody titer aiming at the duck viral hepatitis virus III is more than or equal to 1: 8192;

preferably, in the step (c), the yolk antibody extraction step further comprises: (c6) and (3) sterilization:

sterilizing the yolk antibody concentrated solution;

preferably, in step (c6), the sterilization treatment is to use⁶⁰Co irradiating the concentrated solution;

preferably, in step (c6), the irradiation dose should be 10-15 kGy;

preferably, the concentrated solution is freeze-dried to obtain the yolk antibody freeze-dried powder.

5. A yolk antibody for treating, preventing, slowing down or controlling duck viral hepatitis, which is prepared by the preparation method of any one of claims 1 to 4.

6. A yolk antibody composition for treating, preventing, slowing down or controlling duck viral hepatitis, which takes the yolk antibody prepared by the preparation method of any one of claims 1 to 4 as an immunogen.

7. The yolk antibody composition of claim 6, which comprises the yolk antibody prepared by the preparation method according to any one of claims 1 to 4 and an auxiliary material;

preferably, the auxiliary material is a freeze-drying protective agent;

preferably, the freeze-drying protective agent is a mixture comprising 55-65 parts by weight of trehalose, 15-25 parts by weight of mannitol, 5-15 parts by weight of glycine and 5-15 parts by weight of L-cysteine;

preferably, in the yolk antibody composition, when the dry weight of the auxiliary material is 1g, the immunogen is:

10-30ml of neutralizing antibody titer aiming at the type I duck viral hepatitis virus vaccine strain is more than or equal to 1:8192, or a solution or mixture containing equal amounts of said yolk antibodies; and/or

10-30ml of neutralizing antibody titer aiming at the III type duck viral hepatitis virus vaccine strain is more than or equal to 1:8192, or a solution or mixture containing equal amounts of said yolk antibodies;

preferably, the neutralizing antibody titer is determined by chicken embryo neutralization assay.

8. A method of preparing a yolk antibody composition according to claim 6 or 7, comprising the steps of:

and mixing the yolk antibody with the auxiliary materials to obtain a yolk antibody mixture, thus obtaining the yolk antibody composition.

9. The method of claim 8, further comprising the steps of:

drying the yolk antibody mixture to obtain yolk antibody freeze-dried powder;

preferably, the preparation method further comprises the following steps:

freeze-drying the yolk antibody mixture to obtain yolk antibody freeze-dried powder;

preferably, the lyophilization procedure is:

pre-freezing: placing the yolk antibody mixture at-45 ℃ to-35 ℃ to obtain a first mixture;

sublimation: placing the first mixture at-10 ℃ to-1 ℃ to obtain a second mixture;

desorption and drying: placing the second mixture at 25-30 ℃ to obtain the yolk antibody freeze-dried powder;

preferably, the prefreezing is maintained for 4-8 hours;

preferably, the sublimation is maintained for 30-45 hours;

preferably, the desorption drying is maintained for 2 to 6 hours;

preferably, the transition time from said prefreezing to said sublimating is from 0.8 to 1.2 hours;

preferably, the transition time from sublimation to desorption drying is 0.8 to 1.2 hours;

preferably, the preparation method further comprises the following steps:

diluting the yolk antibody freeze-dried powder with normal saline, PBS or water for injection to obtain a yolk antibody injection;

preferably, in the yolk antibody injection, the neutralizing antibody titer against the duck viral hepatitis type I virus is determined by a chicken embryo neutralization test method and is more than or equal to 1:512, and/or the titer of a neutralizing antibody aiming at the duck viral hepatitis virus III is more than or equal to 1: 512.

10. use of a yolk antibody according to any one of claims 1 to 4, a yolk antibody composition according to claim 5 or claim 6 or claim 7 or a method according to claim 8 or claim 9 in the preparation of a preparation for use alone, in combination with other immunological agents and/or drugs, or as a component of a combined preparation with other immunological agents and/or drugs for the treatment, prevention, alleviation and/or management of duck viral hepatitis type i and/or duck viral hepatitis type iii.

Technical Field

The invention belongs to the field of biological products, relates to a duck virus hepatitis egg yolk antibody and a preparation method thereof, and particularly relates to a divalent egg yolk antibody for duck virus hepatitis I and duck virus hepatitis III and a preparation method thereof.

Background

The domestic duck stock quantity is about 40 hundred million per year. Duck Viral Hepatitis (DVH) is an acute, lethal infectious disease of duckling caused by Duck Hepatitis A Virus (DHAV) characterized by acute onset, hepatomegaly, hemorrhage and high mortality. The ducklings within 21 days of age are mainly infected to cause typical hepatitis symptoms, the death rate of the ducklings is over 80 percent, and the ducklings are spread rapidly and are widely distributed.

In 2008, DHV was renamed to Duck Hepatitis A Virus (DHAV). The DHAV is divided into A, B, C genotypes which respectively correspond to DHAV-1, DHAV-2 and DHAV-3, OIE reports that the type I of Asian main epidemic duck hepatitis and a variant thereof, namely duck hepatitis A virus, are mainly harmful to ducklings within 3 weeks, and have urgent morbidity and high mortality rate, particularly the morbidity and mortality rate of the ducklings within 1-2 weeks are higher, and the mortality rate can reach more than 30%, so that the DHAV is one of important infectious diseases harmful to the duck breeding industry.

The yolk antibody is an antibody extracted from an immunized egg and directed to a specific antigen, and is called yolk immunoglobulin IgG (egg immunoglobulin IgG), abbreviated as IgY, because the yolk contains only IgG-class antibodies. The process of forming the yolk antibody is as follows: when the body is stimulated by external specific antigen (especially inactivated antigen), a series of immune response reactions are induced, B cells are stimulated to differentiate into plasma cells capable of secreting specific antibodies, and a large amount of specific antibodies are secreted into blood. Meanwhile, in the bodies of laying fowls, specific antibody IgG (all subgroups) in blood gradually enters into ovarian follicles and oviducts under the mediation of ovarian IgG receptors and is accumulated in yolk, and due to the accumulation of the yolk, the content of IgG in the yolk is obviously higher than that in serum, while other antibodies (mainly IgM and IgA) in the serum and ovalbumin in the oviducts are mixed into egg white.

The chicken yolk antibody IgY is an immunoglobulin Ig of chicken, and the chicken IgY is functionally equivalent to a mammal IgG, but structurally different and has a typical spatial conformation of the immunoglobulin. The egg yolk antibody has many advantages.

The yolk antibody study started at the end of the 19 th century, 1893, and Klemperer found that yolk is rich in antibodies; experiments by Jukes in 1934 demonstrated that antibodies in hen serum could be transferred to the yolk, thus providing passive immune protection to chicks. Patters et al demonstrated that IgG was selectively transported to the follicle relative to other plasma proteins. In 1969, Leslie and Clem named this antibody IgY.

The antibodies in avian yolk are mainly yolk immunoglobulins, which have many unique advantages compared to serum antibodies: (1) the egg laid by the immunized duck is collected without blood collection, and the egg yolk antibody can be obtained through purification; (2) has good stability, heat resistance and acid resistance, and can still maintain certain activity at normal temperature. And (3) the treatment effect is obvious, the specificity is strong, and the mass production is easy. (4) Safe, no residue, mild and environment-friendly.

Disclosure of Invention

Aiming at the defects of the prior art, the invention discloses a preparation method of a duck virus hepatitis virus egg yolk antibody, wherein the used vaccine strains are an I type LSE strain and an III type QZE strain which are cultured by using a DF-1 cell line, and the egg yolk antibody has good safety and high titer.

The invention provides a preparation method of a yolk antibody for treating, preventing, slowing down or controlling duck viral hepatitis, which comprises the following steps: immunizing an avian body by taking a duck viral hepatitis virus vaccine strain as an immunogen to prepare the egg yolk antibody;

the duck viral hepatitis virus vaccine strain is a duck viral hepatitis virus I vaccine strain and/or a duck viral hepatitis virus III vaccine strain;

the I-type duck viral hepatitis virus vaccine strain is a virus strain with the microbial preservation number of CGMCC No.19696, or a passage virus strain or a mutant virus strain with unchanged clinical pathogenicity and immunogenicity;

the III type duck viral hepatitis virus vaccine strain is a virus strain with the microorganism preservation number of CGMCC No.19695, or a passage virus strain or a mutant virus strain with unchanged clinical pathogenicity and immunogenicity.

In some embodiments, the method of making comprises the steps of:

(a) preparation of the vaccine:

taking the duck viral hepatitis virus vaccine strain I and/or the duck viral hepatitis virus vaccine strain III as an immunogen to prepare a duck viral hepatitis virus vaccine;

(b) preparing high-immunity poultry eggs:

inoculating the duck viral hepatitis virus vaccine to the poultry body to obtain a high-immunity poultry egg;

(c) extraction of yolk antibody:

and extracting yolk antibodies from the hyperimmune eggs.

In some embodiments, in step (a), the duck viral hepatitis virus vaccine comprises the immunogen and a vaccine adjuvant.

Preferably, the duck viral hepatitis virus vaccine is an inactivated vaccine.

In some embodiments, in step (a), the vaccine adjuvant comprises a lipid adjuvant, a first emulsifier, and a second emulsifier.

In some embodiments, in step (a), the lipid-based adjuvant is selected from white oil.

In some embodiments, in step (a), the first emulsifier is selected from span-80.

In some embodiments, in step (a), the second emulsifier is selected from tween-80.

In some embodiments, in step (a), the components of the duck viral hepatitis virus vaccine comprise, in terms of dosage ratios:

the type I duck viral hepatitis virus vaccine strain: type III duck viral hepatitis virus vaccine strain: oil and fat adjuvants: first emulsifier: 750-^7.0ELD₅₀: 750-2250×10^7.0ELD₅₀150-400g, 10-30g, 5-20g (e.g., 800 × 10 g)^7.0ELD₅₀、900×10^7.0 ELD₅₀、1000×10^7.0ELD₅₀、1100×10^7.0ELD₅₀、1200×10^7.0ELD₅₀、1300×10^7.0 ELD₅₀、1400×10^7.0ELD₅₀、1500×10^7.0ELD₅₀、1600×10^7.0ELD₅₀、1700×10^7.0 ELD₅₀、1800×10^7.0ELD₅₀、1900×10^7.0ELD₅₀、2000×10^7.0ELD₅₀、2100×10^7.0 ELD₅₀、2200×10^7.0ELD₅₀：800×10^7.0ELD₅₀、900×10^7.0ELD₅₀、1000×10^7.0ELD₅₀、 1100×10^7.0ELD₅₀、1200×10^7.0ELD₅₀、1300×10^7.0ELD₅₀、1400×10^7.0ELD₅₀、 1500×10^7.0ELD₅₀、1600×10^7.0ELD₅₀、1700×10^7.0ELD₅₀、1800×10^7.0ELD₅₀、 1900×10^7.0ELD₅₀、2000×10^7.0ELD₅₀、2100×10^7.0ELD₅₀、2200×10^7.0ELD₅₀：160g、180g、200g、220g、240g、260g、280g、300g、320g、340g、360g、 380g：12g、14g、16g、18g、20g、22g、24g、26g、28g：6g、8g、10g、12g、 14g、16g、18g)。

In some embodiments, in step (a), the components of the duck viral hepatitis virus vaccine comprise, in terms of dosage ratios:

the type I duck viral hepatitis virus vaccine strain: oil and fat adjuvants: first emulsifier: 4500 × 10 ═ 1500-^7.0ELD₅₀150-400g, 10-30g, 5-20g (e.g., 1700 × 10 g)^7.0ELD₅₀、 1900×10^7.0ELD₅₀、2100×10^7.0ELD₅₀、2300×10^7.0ELD₅₀、2500×10^7.0ELD₅₀、2700×10^7.0ELD₅₀、2900×10^7.0ELD₅₀、3100×10^7.0ELD₅₀、3300×10^7.0ELD₅₀、 3500×10^7.0ELD₅₀、3700×10^7.0ELD₅₀、3900×10^7.0ELD₅₀、4100×10^7.0ELD₅₀、 4300×10^7.0ELD₅₀：160g、180g、200g、220g、240g、260g、280g、300g、320g、 340g、360g、380g：12g、14g、16g、18g、20g、22g、24g、26g、28g：6g、 8g、10g、12g、14g、16g、18g)。

In some embodiments, in step (a), the components of the duck viral hepatitis virus vaccine comprise, in terms of dosage ratios:

type III duck viral hepatitis virus vaccine strain: oil and fat adjuvants: first emulsifier: 4500 × 10 ═ 1500-^7.0ELD₅₀150-400g, 10-30g, 5-20g (e.g., 1700 × 10 g)^7.0ELD₅₀、 1900×10^7.0ELD₅₀、2100×10^7.0ELD₅₀、2300×10^7.0ELD₅₀、2500×10^7.0ELD₅₀、 2700×10^7.0ELD₅₀、2900×10^7.0ELD₅₀、3100×10^7.0ELD₅₀、3300×10^7.0ELD₅₀、 3500×10^7.0ELD₅₀、3700×10^7.0ELD₅₀、3900×10^7.0ELD₅₀、4100×10^7.0ELD₅₀、 4300×10^7.0ELD₅₀：160g、180g、200g、220g、240g、260g、280g、300g、320g、 340g、360g、380g：12g、14g、16g、18g、20g、22g、24g、26g、28g：6g、 8g、10g、12g、14g、16g、18g)。

In some embodiments, in step (a), the ELD of said duck viral hepatitis virus type i vaccine strain and/or said duck viral hepatitis virus type iii vaccine strain₅₀Is calculated according to a Reed-Muench method based on duck embryo culture.

In some embodiments, in step (a), the preparation of the duck viral hepatitis virus vaccine comprises the steps of:

(a1) preparation of oil phase adjuvant:

mixing the oil-fat adjuvant with a first emulsifier to obtain an oil-phase adjuvant;

(a2) preparation of the aqueous phase:

mixing the inactivated duck viral hepatitis virus vaccine strain with a second emulsifier to obtain a water phase;

(a3) mixing:

mixing the oil phase adjuvant with the water phase to obtain the vaccine composition;

in some embodiments, in step (b), the preparation of the high-immunity poultry egg comprises:

(b1) basic inoculation:

inoculating the duck viral hepatitis virus vaccine to poultry in an egg laying period;

(b2) and (3) strengthening inoculation:

inoculating the duck viral hepatitis virus vaccine to the laying-period avian after basal inoculation for 10-28 days (e.g., 12 days, 14 days, 16 days, 18 days, 20 days, 22 days, 24 days, 26 days);

(b3) enhanced inoculation:

and (3) after 10-28 days of boosting inoculation (such as 12 days, 14 days, 16 days, 18 days, 20 days, 22 days, 24 days and 26 days), inoculating the duck viral hepatitis virus vaccine to poultry in an egg producing period to obtain the hyperimmune egg.

In some embodiments, the avian animal is selected from the group consisting of: chicken, duck, goose.

In some embodiments, the egg laying period is 120-400 days old (e.g., 150 days old, 180 days old, 210 days old, 240 days old, 270 days old, 300 days old, 330 days old, 360 days old, 390 days old).

In some embodiments, the basal inoculation is subcutaneous injection containing 1.5-6.0 × 10^7.0ELD₅₀The duck viral hepatitis virus vaccine (2.0 × 10)^7.0ELD₅₀、2.5×10^7.0ELD₅₀、 3.0×10^7.0ELD₅₀、3.5×10^7.0ELD₅₀、4.0×10^7.0ELD₅₀、4.5×10^7.0ELD₅₀、 5.0×10^7.0ELD₅₀、5.5×10^7.0ELD₅₀)。

In some embodiments, the booster vaccination is intramuscular injection containing 3.0-12.0X 10^7.0ELD₅₀ (3.5×10^7.0ELD₅₀、4.0×10^7.0ELD₅₀、4.5×10^7.0ELD₅₀、5.0×10^7.0ELD₅₀、 5.5×10^7.0ELD₅₀、6.0×10^7.0ELD₅₀、6.5×10^7.0ELD₅₀、7.0×10^7.0ELD₅₀、 7.5×10^7.0ELD₅₀、8.0×10^7.0ELD₅₀、8.5×10^7.0ELD₅₀、9.0×10^7.0ELD₅₀、 9.5×10^7.0ELD₅₀、10.0×10^7.0ELD₅₀、10.5×10^7.0ELD₅₀、11.0×10^7.0ELD₅₀、 11.5×10^7.0ELD₅₀) The duck viral hepatitis virus vaccine of the virus.

In some embodiments, the booster vaccination intramuscular injection contains 3.0 to 12.0 x 10^7.0ELD₅₀ (3.5×10^7.0ELD₅₀、4.0×10^7.0ELD₅₀、4.5×10^7.0ELD₅₀、5.0×10^7.0ELD₅₀、 5.5×10^7.0ELD₅₀、6.0×10^7.0ELD₅₀、6.5×10^7.0ELD₅₀、7.0×10^7.0ELD₅₀、 7.5×10^7.0ELD₅₀、8.0×10^7.0ELD₅₀、8.5×10^7.0ELD₅₀、9.0×10^7.0ELD₅₀、 9.5×10^7.0ELD₅₀、10.0×10^7.0ELD₅₀、10.5×10^7.0ELD₅₀、11.0×10^7.0ELD₅₀、 11.5×10^7.0ELD₅₀) The duck viral hepatitis virus vaccine of the virus.

In some embodiments, in step (b), the neutralizing antibody titer against the duck viral hepatitis type I virus in the hyperimmunized avian eggs, as determined by the chick embryo neutralization assay, is greater than or equal to 1:2048 and/or the neutralizing antibody titer against the duck viral hepatitis type III virus is more than or equal to 1: 2048.

in some embodiments, in step (c), the yolk antibody is extracted by:

(c1) separating egg yolk:

obtaining yolk of the high-immunity poultry egg;

(c2) and (3) extraction:

and extracting the yolk to obtain the crude yolk antibody.

In some embodiments, the egg yolk is separated after sterilizing the hyperimmunized egg prior to step (c 1).

In some embodiments, the method of disinfecting the hyperimmunized egg is:

disinfecting the high-immunity poultry eggs by using a new benzalkonium bromide water solution.

In some embodiments, the concentration of the aqueous benzalkonium bromide solution is 0.08-0.12 wt% (e.g., 0.09 wt%, 0.10 wt%, 0.11 wt%).

In some embodiments, the hyperimmune egg is disinfected with an aqueous solution of benzalkonium bromide at a temperature of 35-45 ℃ (e.g., 36 ℃, 37 ℃, 38 ℃, 39 ℃, 40 ℃, 41 ℃, 42 ℃, 43 ℃, 44 ℃).

In some embodiments, the method of disinfecting poultry eggs further comprises:

hermetically fumigating the hyperimmune poultry disinfected with benzalkonium bromide aqueous solution with formalin.

In some embodiments, the time of fumigation ranges from 20 to 40 minutes (e.g., 25 minutes, 30 minutes, 35 minutes).

In some embodiments, in step (c1), the hyperimmunized egg is manually or instrumentally beaten, egg white, blastoderm and frenulum are removed, and the yolk is collected.

In some embodiments, in step (c2), the step of extracting is:

mixing the yolk with acidified water to obtain a yolk mixed solution;

and centrifuging the yolk mixed solution, and collecting supernatant to obtain the crude yolk antibody.

In some embodiments, in step (c2), the acidified water has a pH of 3.5 to 4.5 (e.g., 3.7, 3.9, 4.1, 4.3).

In some embodiments, in step (c2), the volume ratio of the egg yolk to the acidified water is 1:4 to 8 (e.g., 1:5, 1:6, 1: 7).

In some embodiments, in step (c2), the mixing is allowed to stand at 2-5 ℃ (e.g., 3 ℃, 4 ℃) for 3-8 hours (e.g., 4 hours, 5 hours, 6 hours, 7 hours).

In some embodiments, in step (c2), the mixing is performed under stirring conditions.

In some embodiments, in step (c2), the centrifugation is performed at 6000-10000 rpm (e.g., 6500 rpm, 7000 rpm, 7500 rpm, 8000 rpm, 8500 rpm, 9000 rpm, 9500 rpm).

In some embodiments, in step (c2), the centrifugation time is 10-40 minutes (e.g., 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes).

In some embodiments, in step (c), the yolk antibody extraction step further comprises:

(c3) and treating the crude yolk antibody with a clarifying agent to obtain clarified yolk antibody.

In some embodiments, the clarifying agent is n-octanoic acid.

In some embodiments, in step (c3), the treatment time with the clarifying agent is 5 to 10 hours (e.g., 6 hours, 7 hours, 8 hours, 9 hours).

In some embodiments, in step (c3), the clarifying agent is present in the crude egg yolk antibody in an amount of 0.05 to 0.1 v/v% (e.g., 0.06 v/v%, 0.07 v/v%, 0.08 v/v%, 0.09 v/v%).

In some embodiments, in step (c), the yolk antibody extraction step further comprises:

(c4) purification of yolk antibody:

purifying the clarified yolk antibody to obtain the purified yolk antibody.

In some embodiments, in step (c4), the purification step of the yolk antibody is:

and filtering and clarifying the clarified egg yolk antibody.

In some embodiments, in step (c4), the clarifying by filtration is filtration using a 0.5 μm filter cartridge, and the filtrate is taken.

In some embodiments, in step (c4), the step of purifying the yolk antibodies further comprises:

and filtering and sterilizing the filtrate obtained by filtering and clarifying.

In some embodiments, in step (c4), the filter sterilization is filtration using a 0.22 μm filter cartridge to obtain a filtrate.

In some embodiments, in step (c), the yolk antibody extraction step further comprises: (c5) concentrating:

and concentrating the egg yolk antibody solution to obtain an egg yolk antibody concentrated solution.

In some embodiments, the concentration is obtained by concentrating with ultrafiltration membrane having a molecular weight cut-off of 80-120kD (e.g., 90kD, 100kD, 110 kD).

In some embodiments, the yolk antibody concentrate has a neutralizing antibody titer against the duck viral hepatitis type I virus of greater than or equal to 1:8192, and/or the neutralizing antibody titer aiming at the duck viral hepatitis virus III is more than or equal to 1: 8192.

in some embodiments, in step (c), the yolk antibody extraction step further comprises: (c6) sterilization:

and sterilizing the yolk antibody concentrated solution.

In some embodiments, in step (c6), the sterilization treatment is with⁶⁰Co irradiates the concentrated solution.

In some embodiments, in step (c6), the irradiation dose should be 10-15kGy (e.g., 11kGy, 12kGy, 13kGy, 14 kGy).

In some embodiments, the concentrate is lyophilized to obtain a yolk antibody lyophilized powder.

In a second aspect, the invention provides a yolk antibody for treating, preventing, slowing down or controlling duck viral hepatitis, wherein the yolk antibody is prepared by the preparation method of the first aspect of the invention.

In a third aspect, the invention provides a yolk antibody composition for treating, preventing, slowing down or controlling duck viral hepatitis, wherein the yolk antibody composition takes the yolk antibody prepared by the preparation method of the first aspect of the invention as an immunogen.

In some embodiments, the yolk antibody composition comprises the yolk antibody prepared by the preparation method according to the first aspect of the present invention and an auxiliary material.

In some embodiments, the excipient is a lyoprotectant.

In some embodiments, the lyoprotectant is a composition comprising 55-65 parts (e.g., 56, 57, 58, 59, 60, 61, 62, 63, 64) trehalose, 15-25 parts (e.g., 16, 17, 18, 19, 20, 21, 22, 2, 24) mannitol, 5-15 parts (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14) glycine, and 5-15 parts (e.g., 6, 7, 8, 9, 11, 12, 13, 9, 11, 12, 13, 9, 11, 12, 13, 9, 10, 11, 12, 13, 1, or more parts) glycine, 14 parts by weight) of a mixture of L-cysteine.

In some embodiments, in the yolk antibody composition, when the dry weight of the adjuvant is 1g, the immunogen is:

10-30ml (for example, 12ml, 14ml, 16ml, 18ml, 20ml, 22ml, 24ml, 26ml, 28ml) of neutralizing antibody titer against the duck viral hepatitis virus type I vaccine strain is more than or equal to 1:8192, or a solution or mixture containing equal amounts of said yolk antibodies; and/or

10-30ml (for example, 12ml, 14ml, 16ml, 18ml, 20ml, 22ml, 24ml, 26ml, 28ml) of neutralizing antibody titer for the duck virus hepatitis virus type III vaccine strain is more than or equal to 1:8192, or a solution or mixture containing equal amounts of said egg yolk antibodies.

In some embodiments, the neutralizing antibody titer is determined by a chick embryo neutralization assay. In a fourth aspect, the present invention provides a method for preparing a yolk antibody composition according to the third aspect, wherein the method comprises the following steps:

and mixing the yolk antibody with the auxiliary materials to obtain a yolk antibody mixture, thus obtaining the yolk antibody composition.

In some embodiments, the method of making further comprises the steps of:

and drying the yolk antibody mixture to obtain the yolk antibody freeze-dried powder.

In some embodiments, the method of making further comprises the steps of:

and freeze-drying the yolk antibody mixture to obtain the yolk antibody freeze-dried powder.

In some embodiments, the lyophilization procedure is:

pre-freezing: placing the yolk antibody mixture at-45 ℃ to-35 ℃ (such as-44 ℃, -43 ℃, -42 ℃, -41 ℃, -40 ℃, -39 ℃, -38 ℃, -37 ℃, -36 ℃) to obtain a first mixture;

sublimation: placing the first mixture at-10 ℃ to-1 ℃ (such as-9 ℃, -8 ℃, -7 ℃, -6 ℃, -5 ℃, -4 ℃, -3 ℃, -2 ℃) to obtain a second mixture;

desorption and drying: placing the second mixture at 25 deg.C to 30 deg.C (e.g., 26 deg.C, 27 deg.C, 28 deg.C, 29 deg.C, 30 deg.C) to obtain the lyophilized powder of yolk antibody.

In some embodiments, the prefreezing is maintained for 4-8 hours (e.g., 5 hours, 6 hours, 7 hours).

In some embodiments, the sublimation is maintained for 30-45 hours (e.g., 32 hours, 34 hours, 36 hours, 38 hours, 40 hours, 42 hours, 44 hours).

In some embodiments, the desorption drying is maintained for 2-6 hours (e.g., 3 hours, 4 hours, 5 hours).

In some embodiments, the transition time from prefreezing to sublimation is 0.8 to 1.2 hours (e.g., 0.9 hours, 1.0 hours, 1.1 hours).

In some embodiments, the transition time from sublimation to desorption drying is 0.8 to 1.2 hours (e.g., 0.9 hours, 1.0 hours, 1.1 hours).

In some embodiments, the method of making further comprises the steps of:

diluting the yolk antibody freeze-dried powder by using normal saline, PBS or water for injection to obtain a yolk antibody injection.

In some embodiments, the yolk antibody injection has a neutralizing antibody titer against the duck viral hepatitis type I virus of greater than or equal to 1:512, and/or the titer of a neutralizing antibody aiming at the duck viral hepatitis virus III is more than or equal to 1: 512.

in a fifth aspect, the invention provides a preparation method of the first aspect, a yolk antibody of the second aspect, a yolk antibody composition of the third aspect or a preparation method of the fourth aspect, and the use of the preparation method in preparation of a preparation for use alone, in combination with other immune preparations and/or medicaments, or as a component of a compound preparation consisting of other immune preparations and/or medicaments for treating, preventing, slowing and/or controlling duck virus hepatitis type i and/or duck virus hepatitis type iii.

Drawings

FIG. 1 is a technical scheme for preparing the bivalent inactivated vaccine for duck viral hepatitis type I and duck viral hepatitis type III.

FIG. 2 is a technical route for preparing the yolk antibody of the invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention will be described in detail with reference to the accompanying drawings.

Defining:

a passaged or mutant strain with no changes in clinical pathogenicity and immunogenicity:

it is apparent that the virus strains claimed in the present invention are passaged virus strains without mutations belonging to substantially the same virus strains, and that no changes in clinical pathogenicity and immunogenicity mean that no substantial changes are observed, and that differences in animal species, age, sex, health, etc., and predictable systematic errors are passaged virus strains without substantial changes in clinical pathogenicity and immunogenicity due to the assay operating conditions. In addition, minor mutations are inevitably introduced after the virus is passaged many times, and when the mutations occur in non-coding sequence regions or synonymous mutations of coding regions or mutations that do not affect the pathogenicity and immunogenicity of the virus (for example, there may be a connecting amino acid residue between two domains, or a residue of a minor mutation located in a higher structure of a protein that does not affect the pathogenicity or immunogenicity because it is not in contact with immune cells), these minor mutations remain insubstantial mutations and should be regarded as mutant virus strains without changes in clinical pathogenicity and immunogenicity. For example, the avian embryo (e.g., duck embryo) passaged virus strain (or adapted virus strain), the cell (e.g., DF-1 cell line) passaged virus strain (or adapted virus strain), the progeny virus strain which is not sequentially passaged through the avian embryo and the cell, and the progeny virus strain which is crossly passaged through the avian embryo and the cell for many times, which have no substantial changes in clinical pathogenicity and immunogenicity, belong to the passage virus strain or the mutant virus strain having no changes in clinical pathogenicity and immunogenicity.

The invention refers to the current 'Chinese animal pharmacopoeia' which is published by the Chinese agriculture publishing company and is divided into three parts of 'Chinese animal pharmacopoeia' 2015 edition.

Susceptible duck: duck viral hepatitis vaccine is not used in duck farms, and duck viral hepatitis does not occur; duckling hatched by hatching eggs with the duck virus hepatitis neutralizing antibody titer less than 1:4 in the yolk and ducks of different ages in days.

Material

DF-1 cell line, a chicken embryo fibroblast cell line, was purchased from ATCC (ATCC cell line number: CRL-12203)).

The strong virus for testing is a type I LS strain and a type III QZ strain of duck virus hepatitis virus liver tissue virus, which are separated, identified, stored and supplied by Liaoning Yikang biological member GmbH.

White oil: white oil, suonobang usa.

Tween-80: supplied by Shenyang Sanri reagent, Inc.

Span-80: supplied by Shanghai Danzhong pharmaceutical chemical Co., Ltd.

Emulsification equipment: a Frouk high-shear dispersion emulsifier.

FIG. 1 shows the basic technical route of the preparation of the bivalent inactivated vaccine for duck viral hepatitis type I and duck viral hepatitis type III.

Fig. 2 shows a basic technical route for preparing the yolk antibody of the invention.

Example 1 acquisition of Duck viral hepatitis type I vaccine strains

(1) Cases of disease

The disease of a duck group in a domestic duck farm in Liaoning province has the following symptoms: nervous symptoms such as general convulsion, leg spasm, and backward bending of head, etc., typical opisthotonus is shown after death, and the typical pathological changes of the autopsy are hepatomegaly and hemorrhage. After the corpse of the dead duck is subjected to autopsy, the liver is enlarged and has bleeding spots, and the duck viral hepatitis is basically diagnosed.

(2) Pathogen isolation

The diseased duck liver tissue is washed 3 times with sterilized normal saline, and the ratio of the tissue to the tissue is 1 g: adding 10ml of normal saline into the liver tissue, adding the liver tissue into sterilized cold normal saline, putting the liver tissue into a tissue mixer, fully mashing the liver tissue at 10000-12000 r/min, repeatedly freezing and thawing at the temperature of below 15 ℃ below zero for 3 times, collecting filtrate, centrifuging the filtrate at the temperature of 12000r/min and 4 ℃ for 30 minutes, and collecting supernatant to obtain liver tissue liquid.

Taking 0.2ml of liver tissue fluid, inoculating a 14-day-old susceptible duck embryo through an allantoic cavity, incubating for 96 hours at 37 ℃, then aseptically collecting allantoic fluid, centrifuging for 30 minutes at 12000r/min at 4 ℃, and collecting supernatant to obtain virus fluid a containing suspected duck virus hepatitis virus.

(3) Specificity test

Diluting virus liquid a with normal saline 10 times, inoculating 5 susceptible duck embryos of 8-10 days old into allantoic cavities of each dilution, each embryo 0.2ml, placing at 37 ℃ for continuous incubation, discarding dead duck embryos before 24 hours, taking out dead duck embryos at any time after 48-168 hours, and calculating ELD according to Reed-Muench method₅₀. Then diluting virus liquid a to 200ELD with physiological saline₅₀0.1 ml. All subsequent ELDs of the invention₅₀The same ELD was used for all the measurements₅₀And (3) a measuring method.

Mixing the diluted virus liquid a with isovolumetric type I duck viral hepatitis virus specific serum (duck-derived positive serum prepared by Liaoning Yikang biological corporation aiming at the identified type I duck viral hepatitis virus), incubating for 60 minutes at 37 ℃, and inoculating 5 susceptible duck embryos of 8-10 days age into the mixture through allantoic cavities, wherein each embryo is 0.2 ml; incubation at 37 ℃ was observed for 168 hours. The duck embryos in the incubation group are all healthy and alive.

Under parallel conditions, diluted virus solution a and a physiological saline mixture with the same volume are used for inoculating duck embryos and used as a control, and the duck embryos in the control group are all dead. Edema of hind limbs and abdomen of dead embryos, systemic and subcutaneous hemorrhage, pinpoint sized bleeding spots and necrotic foci in the liver, retarded embryonic development.

Therefore, the virus liquid a contains the duck virus hepatitis virus type I.

(4) Molecular diagnostics

Designing a pair of primers near a 5' UTR conserved region sequence according to a gene sequence of the duck virus hepatitis virus type I published on GenBank, wherein the primer sequences are as follows:

F1：5’-AGCACGAGAGACCCTTACG-3’

R1：5’-CCCACAGGCTCTCACTAAAG-3’

the nucleic acid in the virus liquid a was subjected to RT-PCR amplification using primers F1 and R1, the PCR product was sent to Jilin Kuumei bioengineering Co., Ltd for sequencing, and the result was subjected to sequence analysis using DNAStar software, and compared with the gene sequence in NCBI, the sequence having the highest homology with the type I duck viral hepatitis virus VP1 gene was 95.6% homologous, whereby it was confirmed that the virus liquid a contained the type I duck viral hepatitis virus.

The type I duck viral hepatitis virus contained in the virus liquid a is named as type I duck viral hepatitis virus LSE strain (LSE strain for short).

(5) Biological preservation of type I duck viral hepatitis strains

The separated I-type duck viral hepatitis virus LSE strain is submitted to a patent program approved preservation organization for preservation, and the microorganism preservation number of the I-type duck viral hepatitis virus LSE strain is CGMCC No. 19696; the classification is named as: duck viral hepatitis type i virus; the preservation time is as follows: 22/04/2020: the preservation unit is: china general microbiological culture Collection center. The virus strain is named as I-type duck virus hepatitis virus LSE strain, DHAV-1LSE strain, LSE strain and LSE.

Example 2 acquisition of Duck viral hepatitis type III vaccine strains

(1) Cases of disease

The disease of a duck group in a domestic duck farm in Liaoning province has the following symptoms: listlessness, ataxia, drooping wings. Judging according to the symptoms, the suspected duck viral hepatitis is suffered. After autopsy of dead duck, liver swelling, gallbladder swelling and bile color fading, duck viral hepatitis is basically diagnosed.

(2) Pathogen isolation

In the same manner as in the step (2) in example 1, a virus solution b containing a suspected duck viral hepatitis virus was obtained.

(3) Specificity test

The virus solution b was diluted to 200ELD in the same manner as in step (3) of example 1₅₀/0.1ml。

Mixing the diluted virus solution b with isovolumetric type III duck viral hepatitis virus specific serum (duck-derived positive serum prepared by Liaoning Yikang biological corporation aiming at identified type III duck viral hepatitis virus), incubating for 60 minutes at 37 ℃, and inoculating 5 susceptible duck embryos of 8-10 days age into the mixture through allantoic cavities, wherein each embryo is 0.2 ml; incubation at 37 ℃ was observed for 168 hours. The duck embryos in the incubation group are all healthy and alive.

In parallel conditions, diluted virus solution b and a normal saline mixture with the same volume are used for inoculating duck embryos and used as a control, and the duck embryos in the control group are all dead. Edema of dead embryo hind limb and abdomen, systemic and subcutaneous hemorrhage, reddish yellow liver.

Therefore, the virus liquid b is believed to contain the duck virus hepatitis type III virus.

(4) Molecular diagnostics

Designing a pair of primers near a 5' UTR conserved region sequence according to a gene sequence of the duck virus hepatitis virus type III published on GenBank, wherein the primer sequences are as follows:

F3：5’-TGGGCTAATGGTCTTCGT-3’

R3：5’-TTGGGTCTGGTATTGTCTGT-3’

the nucleic acid in the virus liquid b was subjected to RT-PCR amplification using primers F3 and R3, the PCR product was sent to Jilin Cumei bioengineering Co., Ltd for sequencing, and the result was compared with the gene sequence in NCBI by sequence analysis using DNAStar software, which showed 96% homology with the sequence having the highest homology with the type III duck viral hepatitis virus VP1 gene, whereby it was confirmed that the virus liquid b contained the type III duck viral hepatitis virus.

The type III duck viral hepatitis virus contained in the virus liquid b was named as type III duck viral hepatitis virus QZE strain (QZE strain for short).

(5) Biological preservation of type III duck viral hepatitis strains

The invention submits the separated III type duck virus hepatitis virus QZE strain to a patent program approved preservation organization for preservation, and the microorganism preservation number is QZE strain of III type duck virus hepatitis with CGMCC No. 19695; the classification is named as: type III duck viral hepatitis virus; the preservation time is as follows: 22/04/2020: the preservation unit is: china general microbiological culture Collection center. The virus strains are called type III duck viral hepatitis virus QZE strain, DHAV-3QZE strain, QZE strain and QZE strain.

Example 3 cell culture of Duck viral hepatitis type I vaccine strains

(1) Culture of type I duck virus hepatitis virus cell adapted strain

And (2) adopting a limiting dilution purification technology to carry out breeding, inoculating the virus liquid a obtained in the example 1 into a DF-1 cell line for continuous passage, wherein the culture solution is serum-free DMEM-F12 culture medium, selecting progeny with high virus median lethal dose, and culturing for 1-10 generations to obtain the I-type duck viral hepatitis virus DF-1 cell line adapted strain.

The content of different generations of viruses is 10 by determination^7.0-8.5ELD₅₀/0.2ml。

(2) Seed lot for establishing virus seeds of duck virus hepatitis virus type I

Inoculating the DF-1 cell line adapted strain of the duck viral hepatitis virus type I obtained in the step 1) with DF-1 cells, wherein the culture solution is as follows: serum-free DMEM-F12 culture medium, subculturing to 10 th generation by using cell culture triangular shake flask, determining virus content in cell culture solution, and selecting virus seeds of each generation with virus content not less than 10^7.5ELD₅₀Passage or preservation at 0.2 ml. Defatted milk jelly prepared from 5% sucroseA dry protective agent (the solvent is water, the concentration of sucrose is 5 w/v%, the concentration of skimmed milk powder is 10 w/v%) and each generation of virus culture solution are mixed according to the weight ratio of 1:1 volume ratio, subpackaging, freeze-drying and storing at-70 ℃. The sterility test is qualified (the method and the qualification standard refer to the current Chinese veterinary pharmacopoeia, TG and TSB culture medium for detection), the specificity test is qualified (the method and the qualification standard are the same as the step (3) in the example 1), and the virus content test is qualified (more than or equal to 10)^7.5ELD₅₀0.2ml) and the virus of the foreign source is qualified by inspection (the method and the qualified standard refer to the current Chinese veterinary pharmacopoeia), the F2-F4 generation virus is used as the original virus seed, wherein the F5-F7 generation virus is used as the basic virus seed, and the F8-F10 generation virus is used as the production virus seed; determining the multiplicity of infection (MOI) of the virus against DF-1 cells: specifically, the virus content of the F8-F10 virus seeds was measured by 1 ELD₅₀The virus content proliferated after inoculating 10000, 1000, 100, 10 and 1 cells is more than or equal to 10^7.5ELD₅₀The MOI of DF-1 cells inoculated with F8-F10 virus seeds is determined to be 0.001-0.1 according to a standard of 0.2 ml. Determination of Virus content (ELD) of Virus production seed Virus of generations F8-F10 Using Duck embryos₅₀Half lethal amount of duck embryo) of more than or equal to 10^7.5ELD₅₀And the standard of 0.2ml determines that F8-F10 generation virus seeds are inoculated on DF-1 cells, and the inoculation is carried out according to the volume ratio of virus liquid to cell culture liquid of 1/1000-5/1000 in each passage.

(3) Preparation of cell venom of duck viral hepatitis virus type I

DF-1 cells were cultured in full suspension in a 14L bioreactor using a medium consisting of: serum-free DMEM-F12 culture medium, wherein the culture condition is that the temperature is 35-36 ℃, and the pH value is 7.2-7.5; the rotation speed is 130-^7.5ELD₅₀0.2ml, the inoculation amount is 1ml, the culture is continued at 35-37 ℃, after 96-144 hours, the cell venom is harvested when the cell activity is reduced to be below 40 percent, and the virus content of the cell venom is 10^7.4ELD₅₀/0.2ml。

Example 4 cell culture of Duck viral hepatitis type III vaccine strains

(1) Culture of type III duck virus hepatitis virus cell adapted strain

The virus solution b obtained in example 2 was cultured by the same method and criteria as in step (1) of example 3, and 1-10 generations were cultured to obtain a DF-1 cell line-adapted strain of duck viral hepatitis type III virus.

The content of different generations of viruses is 10 by determination^7.1-8.7ELD₅₀/0.2ml。

(2) Establishment of seed lot of III type duck virus hepatitis virus

Inoculating DF-1 cells to the adaptive strain of the III type duck viral hepatitis virus DF-1 cell line obtained in the step 1), wherein the culture solution is as follows: serum-free DMEM-F12 culture medium, subculturing to the 15 th generation by using a cell culture triangular shake flask, determining the virus content in the cell culture solution, and selecting the virus content of each generation of virus seeds to be more than or equal to 10^7.5ELD₅₀Passage or preservation at 0.2 ml. Using sucrose skim milk freeze-drying protective agent and virus culture solution of each generation in a ratio of 1:1 volume ratio, subpackaging, freeze-drying and storing at-70 ℃. The sterility test is qualified (the method and the qualification standard refer to the current Chinese veterinary pharmacopoeia, TG and TSB culture medium for detection), the specificity test is qualified (the method and the qualification standard are the same as the step (3) of the example 2), and the virus content test is qualified (more than or equal to 10)^7.5ELD₅₀0.2ml) and the virus of the foreign source is qualified by inspection (the method and the qualified standard refer to the current Chinese veterinary pharmacopoeia), the F7-F15 generation virus is used as the original virus seed, wherein the F7-F9 generation virus is used as the basic virus seed, and the F10-F12 generation virus is used as the production virus seed; determining the multiplicity of infection (MOI) of the virus against DF-1 cells: specifically, after obtaining the F13-F15 generation 3 type QZE strain as the production seed, determining the virus infection complex number, measuring the virus content of the F13-F15 generation production seed, and carrying out 1 ELD₅₀The virus content proliferated after inoculating 10000, 1000, 100, 10 and 1 cells is more than or equal to 10^7.5ELD₅₀The MOI of DF-1 cells inoculated with F13-F15 virus seeds is determined to be 0.001-0.1 according to a standard of 0.2 ml. Determination of Virus content (ELD) of Virus production seed Virus of generations F13-F15 Using Duck embryos₅₀Half lethal amount of duck embryo) of more than or equal to 10^7.5ELD₅₀0.2ml standard, F13-F15 toxin generations are determinedAnd inoculating DF-1 cells, wherein the inoculation is carried out according to the volume ratio of virus liquid to cell culture liquid of 1/1000-5/1000 in each passage.

(3) Preparation of cell venom of duck viral hepatitis type III virus

DF-1 cells were cultured in full suspension in a 14L bioreactor using a medium consisting of: serum-free DMEM-F12 culture medium, wherein the culture condition is that the temperature is 35-36 ℃, and the pH value is 7.2-7.5; the rotation speed is 130-150rpm, when the DF-1 cell number in the reactor reaches 400-1000 ten thousand, the F13-F15 generation virus seeds cultured by the seed batch are inoculated, the content of the virus seeds is 10^7.5ELD₅₀0.2ml, the inoculation amount is 1ml, the culture is continued at 35-37 ℃, after 96-144 hours, the cell venom is harvested when the cell activity is reduced to be below 40 percent, and the virus content of the cell venom is 10^7.7ELD₅₀/0.2ml。

Example 5 preparation of Duck viral hepatitis vaccine

(1) Inactivation of viruses

(i) Inactivating duck viral hepatitis type I virus:

and (3) repeatedly freezing and thawing the LSE strain cell venom obtained in the example 3 for 3 times, filtering and sterilizing the cell venom by adopting a plurality of layers of sterile gauze, adding an inactivating agent (formaldehyde water solution with the concentration of 37%) which is 0.2 percent of the total amount (V/V) of the cell venom, and oscillating and inactivating the cell venom for 24 hours at the temperature of 36 ℃ to obtain the LSE strain inactivated virus fluid.

And (3) finished product inspection of inactivated viruses: inoculating the LSE strain inactivated virus solution into 10 susceptible duck embryos in a CAM (chorioallantoic membrane) way at a dose of 0.2ml per embryo, observing for 168 hours, judging whether duck virus hepatitis specific lesion or dead duck embryos exist, and judging that the virus solution is completely inactivated; meanwhile, LSE strain inactivated virus liquid is taken and inoculated with TG and TSB culture media for bacterial examination respectively, no bacteria grow after 10 days of observation, and the inactivated virus liquid is judged to have no bacterial pollution.

(ii) Inactivating duck viral hepatitis type III virus:

a QZE inactivated virus solution was obtained by the same method as in (i) of step (1) of example 5 using the QZE strain cell virus solution obtained in example 4.

And (3) finished product inspection of inactivated viruses: the obtained QZE inactivated virus solution was collected and judged to be free from bacterial contamination by the same method as in (i) of step (1) of example 5.

(2) Preparing an oil phase adjuvant:

mixing 95 parts by volume of white oil and 5 parts by volume of span-80, uniformly stirring at 100 ℃, carrying out high-pressure sterilization at 116 ℃, and cooling to obtain the oil phase adjuvant for later use.

(3) Preparation of an aqueous phase:

(i) preparation of a water phase of duck viral hepatitis virus type I:

and fully shaking and uniformly mixing 94 parts by volume of the LSE strain inactivated virus solution and 6 parts by volume of sterilized Tween-80 to obtain an LSE strain water phase.

(ii) Preparing a III type duck viral hepatitis virus aqueous phase:

and mixing 94 parts by volume of the QZE inactivated virus solution and 6 parts by volume of sterilized Tween-80, and sufficiently shaking and uniformly mixing to obtain QZE aqueous phases.

(4) Preparation of the vaccine:

(i) preparation of type I duck viral hepatitis virus vaccine:

3 parts by volume of the oil phase adjuvant was poured into an emulsification tank and stirred, and 2 parts by volume of the LSE strain water phase was slowly added thereto and sufficiently stirred and emulsified. A sterile 1% thimerosal solution was added to a final concentration of 0.01 w/v% prior to termination of emulsification. Obtaining the LSE strain vaccine (LSE strain vaccine for short) of the duck viral hepatitis virus I.

(ii) Preparing a type III duck viral hepatitis virus vaccine:

3 parts by volume of the oil phase adjuvant was poured into an emulsification vessel and stirred, and 2 parts by volume of the QZE strain aqueous phase was slowly added thereto and sufficiently stirred and emulsified. A sterile 1% thimerosal solution was added to a final concentration of 0.01 w/v% prior to termination of emulsification. Obtain the QZE-strain vaccine (QZE-strain vaccine for short) of the III-type duck viral hepatitis virus.

(iii) Preparing a bivalent duck viral hepatitis virus vaccine:

3 parts by volume of the oil phase adjuvant was poured into an emulsification vessel and stirred, and 1 part by volume of the LSE strain aqueous phase and 1 part by volume of the QZE strain aqueous phase were slowly added thereto and sufficiently stirred and emulsified. A sterile 1% thimerosal solution was added to a final concentration of 0.01 w/v% prior to termination of emulsification. Obtaining bivalent inactivated vaccines (bivalent vaccine for short) of I type duck viral hepatitis virus LSE strain and III type duck viral hepatitis virus QZE strain.

Example 6 preparation of lyophilized yolk antibody

(1) Selection of layers

The commercial layer chicken meets the following conditions.

No avian leukemia and reticuloendotheliosis: sampling blood according to 0.5 percent of chicken flocks, respectively detecting antibodies, and obtaining negative results.

The positive rate of pullorum disease and mycoplasma gallisepticum infection is less than or equal to 0.1 percent by detecting according to NY/T536-2002 'diagnosis technology of typhoid and pullorum disease' and NY/T553-2002 'diagnosis technology of mycoplasma gallisepticum disease'.

The laying hens should have the production performance of commercial laying hens.

Chicken feeding management: the construction of the chicken farm meets the requirements of veterinary health and epidemic prevention specifications. The chicken farm should leave the traffic road for more than 200 meters, and the entrance and exit roads should be separated. The material and the manure channel in the field are separated. The inlet and outlet of the chicken farm are provided with a disinfection pond. Isolation belts should be arranged in brooding houses and adult chicken houses. In addition, chicken farms are provided with manure treatment facilities. And implementing a full-in full-out system. The drinking water of the chicken farm should reach the sanitary standard. The feeding personnel are sanitary and healthy.

And (3) chicken group epidemic disease prevention and treatment: timely inoculating chicken Newcastle disease, avian influenza, chicken Marek's disease, chicken infectious bronchitis, chicken infectious rhinitis, chicken egg drop syndrome (EDS-76) and chicken colibacillosis vaccine according to a scientific immunization program. Antibacterial and anticoccidial drugs are added into the feed according to the conventional method to prevent bacterial and coccidian infection.

(2) Immunization procedure

(a) Basic inoculation: a120-day-old commercial layer was injected subcutaneously with 1ml of the bivalent vaccine prepared in example 5 per neck.

(b) And (3) strengthening inoculation: the 2 nd vaccination was carried out 14 days after the basal vaccination, and each chicken was injected intramuscularly in the chest with 2ml of the bivalent vaccine prepared in example 5.

(c) Enhanced inoculation: the 3 rd vaccination was carried out 14 days after the 2 nd vaccination, and 2ml of the bivalent vaccine prepared in example 5 was injected intramuscularly into each chicken leg.

(d) Maintaining inoculation: at 3-4 months after the booster inoculation, when the egg yolk of the chicken-produced eggs has a marginal 1:2048 titer against the neutralizing antibody of the type I or type III duck viral hepatitis virus, the inoculation is maintained for 1 time, and 2ml of the bivalent vaccine prepared in example 5 is injected intramuscularly in each chest.

A neutralization test method of duck viral hepatitis virus type I comprises the following steps: mixing high immunity egg with normal saline 1:3(V/V), extracting supernatant with chloroform of equal volume, diluting the supernatant 2 times, and mixing with I type duck hepatitis virus LSE strain virus liquid (200 ELD) at 9 dilutions of 1:4, 1:16, 1:32, 1:64, 1:128, 1:256, 1:512, 1:1024 and 1:2048₅₀/0, lml) are mixed in equal volume, the mixture is placed at 37 ℃ for neutralization for 1 hour, 5 SPF chick embryos of 10 days old are inoculated in each dilution allantoic cavity, and each embryo is 0.2 ml; setting 5 virus control, inoculating 0.2ml of mixed solution of virus treated under the same condition and normal saline for each virus; blank control 5, each embryo injected with 0.2ml of physiological saline. Incubation was continued at 37 ℃ for 168 hours, and chick embryo death was recorded for each group. Discarding the dead chick embryos 48 hours before, taking out the chick embryos 48-168 hours after, and calculating half protection amount (PD) of the chick embryos₅₀). The highest dilution factor capable of protecting 50 percent of chick embryos is the neutralizing titer of the antibody. This method is called chick embryo neutralization test.

The neutralizing test method of the duck virus hepatitis virus III comprises the following steps: the virus solution is III type duck hepatitis virus III type QZE virus solution (200 ELD)₅₀Lml), other steps and methods are the same as those of the neutralization test of the duck viral hepatitis virus type I.

(3) Preparation of yolk antibody

(a) Egg collection:

on 10-14 days after the 3 rd immunization, the yolk of the eggs from the hyperimmune chickens was sampled and assayed for the titer of neutralizing antibodies against type I and type III duck viral hepatitis viruses (method described above for neutralization test of chicken embryos). After mixing the high-immunity eggs with normal saline at a ratio of 1:3(V/V), extracting supernatant by using chloroform with the same volume, wherein the titer of neutralizing antibodies is more than or equal to 1: above 2048, the product is qualified. Collecting eggs, and storing at 10-15 deg.C for no more than 10 days.

(b) Egg shell disinfection:

the eggs qualified in the sampling test are soaked in 0. lwt% benzalkonium bromide water solution at 42 ℃ for 15 minutes for disinfection. Screening out egg shells with serious pollution, washing with 70% (v/v) ethanol, soaking in 0. lwt% benzalkonium bromide water solution at 42 deg.C for 15 min, and sterilizing for 1 time. Finally, the mixture was fumigated in formalin for 30 minutes in a closed manner.

(c) Breaking eggs and separating yolk:

and (5) beating eggs by using a yolk separator. The egg white, blastoderm and frenulum were removed sufficiently and the egg yolk was collected.

(d) And (3) extraction:

and (3) adding 6 times volume of acidified water (water for injection with pH value adjusted to 4.2 by hydrochloric acid) of the yolk obtained in the step (c) into a stainless steel tank, cooling to 4 ℃, adding the yolk obtained in the step (c), stirring while adding, standing for 5 hours at 4 ℃, and after acidification is finished, centrifuging for 15 minutes by using a low-temperature continuous centrifuge at 8000 rpm to obtain a supernatant.

(e) Clarification:

the supernatant was transferred to another reaction tank. Adding n-octanoic acid with final concentration of 0.3 v/v% into the supernatant, and standing at room temperature for 5-10 hr.

The function of this step is: crude protein and residue in yolk are separated and removed.

(f) And (3) filtering:

filtering and clarifying with 0.5 μm cylindrical filter core, collecting filtrate, filtering the filtrate with 0.22 μm cylindrical filter core, and sterilizing to obtain filtrate.

(g) Concentration:

concentrating the filtrate with ultrafiltration membrane with molecular weight cutoff of 100KD by 5-10 times to obtain concentrated solution.

(h) Inspection of semi-finished product

And (3) detecting the titer of the antibody: and (3) adopting the chick embryo neutralization test method, diluting the concentrated solution obtained in the step (g) by 2 times in series, and measuring the neutralizing antibody of the duck viral hepatitis virus type I or III, wherein the antibody titer is more than or equal to 1: 8192.

(i) Irradiation of radiation: using the concentrated solution⁶⁰Co irradiation with a dose of 12kGy。

The function of this step is sterilization.

(j) Preparing and subpackaging an antibody: according to the titer of the neutralizing antibody of the semi-finished product, the sterile mixed batch ensures that the titer of the neutralizing antibody is more than or equal to 1: 8192.

Mixing the concentrated solution of the previous step (g) with a freeze-drying protective agent according to a volume ratio of 95 ml: mixing 5g, shaking, and packaging into 5 ml/bottle.

The freeze-drying protective agent comprises: a mixture of 60 wt% trehalose, 20 wt% mannitol, 10 wt% glycine and 10 wt% L-cysteine, sterilized and used.

And (4) quickly freezing and drying in vacuum after subpackaging. The freeze-drying process comprises the following steps:

pre-freezing: the product was put in a box at room temperature, and the product was cooled to-40 ℃ for 1 hour and maintained for 6 hours.

A sublimation stage: the preparation was raised from-40 ℃ to-5 ℃ for 1 hour and maintained for 38 hours.

A desorption drying stage: the preparation was raised from-5 ℃ to 28 ℃ for 1 hour and maintained for 4 hours, and lyophilized for a total period of 48 hours.

The produced yolk antibody is called bivalent yolk antibody.

Example 6 yolk antibody Final product assay

3 batches of the bivalent yolk antibody were independently prepared according to the method of example 5, and the following tests were performed.

(a) The characteristics are as follows: the 3 batches of freeze-dried bivalent yolk antibodies are yellowish spongy loose lumps and are easy to separate from the bottle wall, and after being dissolved by the sterilized normal saline, the 3 batches of freeze-dried bivalent yolk antibodies are yellowish liquid.

(b) And (4) sterile inspection: the test is carried out according to the appendix of the current Chinese veterinary pharmacopoeia.

As a result: TG and TSB culture medium were inoculated with 3 batches of bivalent yolk antibody, respectively, and all showed no bacterial and mold contamination.

(c) And (3) mycoplasma test: the test is carried out according to the method in the appendix of the current Chinese veterinary pharmacopoeia.

As a result: no mycoplasma colony appears in 3 batches of bivalent yolk antibody cultured on agar solid plates, and the pH values of liquid culture mediums are 7.48, 7.45 and 7.47 respectively, which shows that no mycoplasma pollution exists.

(d) And (3) exogenous virus inspection: according to the requirements of poultry-derived cells and products thereof and other poultry-derived products inspection in the existing 'foreign virus inspection method' appendix 'of Chinese veterinary pharmacopoeia', the detection is carried out by adopting a chick embryo inspection method and a cell inspection method, and 3 batches of bivalent egg yolk antibodies have no foreign virus pollution.

Example 7 yolk antibody safety test

3 batches of bivalent yolk antibodies, respectively, were independently prepared according to the method of example 5, and counted as 01, 02, and 03 batches, respectively. Randomly drawing 5 bottles of the yolk antibody into one unit for each batch, diluting the bivalent yolk antibody into 80ml by using sterilized normal saline for each bottle, ensuring that the test is carried out by adopting the chick embryo neutralization test method, wherein the titer of the two kinds of neutralizing antibodies is more than or equal to 1:512, and each ml is 1 feather, and uniformly mixing 5 bottles of bivalent yolk antibody injection for use. The following security check is performed.

The duck for test: cherry valley duck. Duck viral hepatitis vaccine is not used in duck farms, and duck viral hepatitis does not occur; duckling hatched by hatching eggs with the duck virus hepatitis neutralizing antibody titer less than 1:4 in the yolk and ducks of different ages in days.

Feeding and managing ducks: the control ducks and the test ducks of each group are independently raised in a shed and are distinguished by adopting the dyeing marks of different parts of the ducks of each group. The animals were kept by the test person and observed daily.

Mice for the test: and (4) cleaning grade.

And (3) feeding and managing the mice: the mice in the control group and each test group were individually housed and differentiated by labeling the cage walls with a marker. The animals were kept by the test person and observed daily.

The duckling reference health examination standard: the weight meets the requirements of the variety, and the population is regular; covering the umbilicus with velveteen, and tightening and drying; the abdomen is soft, the yolk is well absorbed, and the feathers are clean and glossy; the product is full of vitality, good in spirit and sensitive in response; when the hand is held, the spring is felt, struggling forcefully and sounding greatly.

The standard of reference health examination of breeding ducks: the duck head is medium in size, the neck is thin and long, eyes are bright and have spirit, the beak is long and straight, the body is long and back is wide, the chest is deep and the abdomen is round, the back is wide, the pubis is open, the feathers are compact, the two wings are tightly attached to the body, the feet are slightly short, the webs are large and thick, the duck is healthy and firm, and the body fat is moderate.

(1) Minimum day-old different route single dose injection safety test

80 healthy cherry valley ducks of 1 day old are taken and randomly divided into 8 groups, each group comprises 10 healthy cherry valley ducks, the male ducks and the female ducks are half numbered and respectively called and recorded as ducklings. Each batch of divalent yolk antibody injection is injected with 10 healthy and susceptible ducklings of 1 day old by muscle and subcutaneous injection, and each ducklings is 0.5 ml. Meanwhile, 20 ducklings with age of 1 day in the control group are injected with 10 ducklings subcutaneously and intramuscularly respectively, and each ducklings is injected with 0.5ml of physiological saline. Isolated breeding and observation are carried out for 10 days under the same conditions, and the results are recorded.

As a result: in the experimental observation period, all the ducklings have good mental states, no abnormal behavior activities, normal food intake, drinking water and excrement, and no obvious difference from a control group. During the test period, all the ducklings used for the test are healthy and alive, have no adverse reaction, have no obvious difference with a control group and have good safety. The average daily gain is not significantly different from the control group; the average body weight was not significantly different from the control group; the ducklings injected have no local reaction after being pressed, and all ducklings have no red swelling, induration or other local reactions after being injected with the vaccine; the liver, spleen, kidney, heart and gall bladder are observed by systematic caesarean section, and the results show no obvious change compared with the ducklings in a control group.

(2) Single dose repeat injection safety test

80 healthy cherry valley ducks of 4 days old are taken and randomly divided into 8 groups, each group comprises 10 healthy cherry valley ducks, the male ducks and the female ducks are half numbered and respectively called and recorded as ducklings. Each batch of bivalent egg yolk antibody injection is injected with 10 healthy and susceptible ducklings of 4 days old, 0.5 ml/ducklings, intramuscularly and subcutaneously. The injection is repeated 1 time at 48 hours after the injection, 0.5 ml/injection, and each injection is not in the same position. Meanwhile, 20 ducklings with the age of 4 days in a control group are injected with 10 ducklings subcutaneously and intramuscularly respectively, and each ducklings is injected with 0.5ml of physiological saline. Isolated breeding and observation are carried out for 10 days under the same conditions, and the results are recorded.

As a result: in the experimental observation period, all the ducklings have good mental states, no abnormal behavior activities, normal food intake, drinking water and excrement, and no obvious difference from a control group. During the test period, all the ducklings used for the test are healthy and alive, have no adverse reaction, have no obvious difference with a control group and have good safety. The average daily gain is not significantly different from the control group; the average body weight was not significantly different from the control group; the ducklings injected have no local reaction after being pressed, and all ducklings have no red swelling, induration or other local reactions after being injected with the vaccine; the liver, spleen, kidney, heart and gall bladder are observed by systematic caesarean section, and the results show no obvious change compared with the ducklings in a control group.

(3) Overdose different route injection safety test

80 healthy cherry valley ducks of 4 days old are taken and randomly divided into 8 groups, each group comprises 10 healthy cherry valley ducks, the male ducks and the female ducks are half numbered and respectively called and recorded as ducklings. Each batch of divalent yolk antibody injection is injected with 10 healthy and susceptible ducklings of 4 days old, 2.0 ml/ducklings, intramuscularly and subcutaneously. Meanwhile, 20 ducklings with the age of 4 days in a control group are injected with 10 ducklings subcutaneously and intramuscularly respectively, and each ducklings is injected with 2.0ml of physiological saline. Isolated breeding and observation are carried out for 10 days under the same conditions, and the results are recorded.

As a result: in the experimental observation period, all the ducklings have good mental states, no abnormal behavior activities, normal food intake, drinking water and excrement, and no obvious difference from a control group. During the test period, all the ducklings used for the test are healthy and alive, have no adverse reaction, have no obvious difference with a control group and have good safety. The average daily gain is not significantly different from the control group; the average body weight was not significantly different from the control group; the ducklings injected have no local reaction after being pressed, and all ducklings have no red swelling, induration or other local reactions after being injected with the vaccine; the liver, spleen, kidney, heart and gall bladder are observed by systematic caesarean section, and the results show no obvious change compared with the ducklings in a control group.

(4) Safety test for different varieties of ducks

Taking 40 healthy cherry valley ducks, sheldrakes and Beijing ducks of 4 days old, randomly dividing the healthy cherry valley ducks, sheldrakes and Beijing ducks into 4 groups, 10 ducks in each group are numbered in each half of the male and female parts, and respectively weighing and recording the weights of the ducklings. For each variety of duck, 10 healthy and susceptible ducklings of 4 days old are respectively injected intramuscularly and subcutaneously by using 01 batches of divalent egg yolk antibody injection, and each ducklings is 2 ml. Meanwhile, 20 ducklings with the age of 4 days in a control group are injected with 10 ducklings subcutaneously and intramuscularly respectively, and each ducklings is injected with 2ml of physiological saline. Isolated breeding and observation are carried out for 10 days under the same conditions, and the results are recorded.

As a result: in the experimental observation period, all the ducklings have good mental states, no abnormal behavior activities, normal food intake, drinking water and excrement, and no obvious difference from a control group. During the test period, all the ducklings used for the test are healthy and alive, have no adverse reaction, have no obvious difference with a control group and have good safety. The average daily gain is not significantly different from the control group; the average body weight was not significantly different from the control group; the ducklings injected have no local reaction after being pressed, and all ducklings have no red swelling, induration or other local reactions after being injected with the vaccine; the liver, spleen, kidney, heart and gall bladder are observed by systematic caesarean section, and the results show no obvious change compared with the ducklings in a control group.

(5) Influence on duck production performance

40 healthy Muscovy ducks of 210 days old are taken, randomly divided into 4 groups, 10 female ducks in each group are numbered, the feeding of the female ducks is observed, and the egg laying number is recorded. Each batch of divalent yolk antibody injection is injected intramuscularly with 10 healthy and susceptible ducklings of 210 days old, 2.0 ml/ducklings. Meanwhile, 10 ducklings with the age of 210 days in a control group are provided, and 0.5ml of physiological saline is injected into each muscle. Isolated breeding and observation are carried out for 10 days under the same conditions, and the results are recorded.

As a result: in the experimental observation period, all the breeding ducks are in good mental state, the behavior and the activity are not abnormal, and the eating, drinking and excrement are normal and have no obvious difference from the control group. During the test period, all the tested breeding ducks are healthy and alive without any adverse reaction and obvious difference with a control group, and the safety is good. The average daily egg production number is not obviously different from that of a control group; the injected breeding ducks have no local reaction after being pressed, and the breeding ducks have no red swelling, induration or other local reactions after being injected with the vaccine.

(6) Safety test for non-target animal mice

Each batch of bivalent yolk antibody injection is injected with 10 clean-grade mice of 18-22 g, 0.5ml each. At the same time, 10 healthy controls were provided, and each control was injected with 0.5ml of physiological saline. Isolated breeding and observation under the same condition for 10 days.

As a result: in the experimental observation period, all mice have good mental state, no abnormal behavior activity, normal food intake, drinking water and feces, and no obvious difference from the control group. During the test period, all the white mice used for the test are healthy and alive, have no adverse reaction, have no obvious difference with a control group, and have good safety. The injected mice have no local reaction after being pressed, and all the mice have no red swelling, induration and other visible local reactions after being injected with the vaccine; the liver, spleen, kidney, small intestine and gallbladder were observed by autopsy, and no significant change was observed compared with the control group of mice.

Example 8 yolk antibody protective efficacy test

The yolk antibody injection used was the same as batch 01 of example 7.

(1) Duck viral hepatitis lyophilized yolk antibody different use approaches toxicity counteracting protection test

Taking 90 healthy cherry valley ducks of 1 day old, randomly dividing the healthy cherry valley ducks into 9 groups, 10 ducks in each group, numbering the male and female halves, and respectively weighing and recording the weights of the ducklings. Injecting 1.0ml bivalent yolk antibody injection into 1 group of muscle, and injecting 0.5ml virulent strain of type I LS strain (containing 100 LD) of duck viral hepatitis virus into muscle after 24 hr₅₀) (ii) a Intramuscular injection of 1.0ml bivalent yolk antibody injection, 24h later intramuscular injection of 0.5ml virulent strain of duck viral hepatitis virus type III QZ (containing 100 LD)₅₀) (ii) a Subcutaneously injecting 1.0ml bivalent yolk antibody injection, and intramuscularly injecting 0.5ml (containing 100 LD) virulent strain of type I LS of duck viral hepatitis virus (DHV) 24h later₅₀) (ii) a Subcutaneously injecting 1.0ml bivalent yolk antibody injection, and intramuscularly injecting 0.5ml virulent strain (containing 100 LD) of duck viral hepatitis virus type III QZ strain after 24h₅₀). Inoculating 4 groups of duck with normal saline, wherein 2 groups of duck virus hepatitis virus type I LS strain is injected into muscle with 0.5ml (containing 100 LD) after 24 hr of normal saline is injected into muscle₅₀) And 0.5ml of virulent virus of duck virus hepatitis virus type III QZ strain (containing 100 LD)₅₀) And another 2 groups of duck viral hepatitis virus type I LS strain virulent drugs 0.5ml (containing 100 LD) are respectively injected into the muscle 24h after the normal saline is injected subcutaneously₅₀) And 0.5ml of virulent virus of duck virus hepatitis virus type III QZ strain (containing 100 LD)₅₀). Taking 1 group of ducks as a control, not making injection, not makingCounteracting toxic substances, and feeding all ducks under the same condition. The results are shown in table 1 below:

TABLE 1 Duck viral hepatitis lyophilized yolk antibody different use path toxicity counteracting protection test and results thereof

Therefore, the bivalent egg yolk antibody of the invention has good prevention effect on I type and III type duck viral hepatitis viruses, and can be used clinically.

(2) Duck viral hepatitis lyophilized yolk antibody minimum prophylactic dose test

Respectively injecting 10 Beijing ducks of different ages in days into the above 01 batches of divalent yolk antibody injection or normal saline with different dosages, and subcutaneously injecting the strong virus (content 200 LD) of type I LS strain of duck viral hepatitis virus 24h later₅₀Perml) or virulent virus of duck virus hepatitis virus type III QZ strain (content 200 LD)₅₀Per ml), 10 animals were taken without injection of any substance and without toxicity, and used as controls. Isolated breeding and observation under the same condition for 10 days. And recording the morbidity and mortality of the ducklings at each dose. See table 2 for operating parameters and results.

TABLE 2 Duck viral hepatitis lyophilized yolk antibody minimum preventive dose test and results thereof

Therefore, the bivalent egg yolk antibody of the invention has good prevention effect on I type and III type duck viral hepatitis viruses, and can be used clinically.

(3) Duck viral hepatitis lyophilized yolk antibody minimum dose treatment test

120 cherry valley ducks of 1 day old are taken and injected with 0.5ml duck virus for each subcutaneous injectionHepatitis virus type I LS strain strong poison (content 200 LD)₅₀In ml). After 48 hours post challenge, 94 ducks had symptoms: cachexia, dull movement and closure of both eyes, 80 of them were divided into 4 groups of 20 individuals, and 0.3ml, 0.5ml, 1.0ml and 1.5ml of the above 01 batches of the bivalent yolk antibody injection were injected subcutaneously, respectively. The rest 14 ducks are used as a blank control group without any treatment, are kept in isolation under the same condition and observed for 10 days, and the morbidity and mortality results of the ducks are recorded.

120 cherry valley ducks of 1 day old are taken, and 0.5ml of duck virus hepatitis virus type III QZ strain virulent virus (content 200LD50/ml) is injected into each duck subcutaneously. After 48 hours post challenge, 98 ducks had symptoms: listlessness, dull movement, drooping wings. 80 of the two yolk antibodies were divided into 4 groups, and 20 of the two yolk antibodies were injected subcutaneously with 0.3ml, 0.5ml, 1.0ml and 1.5ml of the above 01 batches of the bivalent yolk antibody injection, respectively. The rest 18 ducks are used as a blank control group without any treatment, are kept in isolation under the same condition for 10 days, and the results of the diseases and the deaths of the ducks are recorded.

See table 3 for specific results.

TABLE 3 Freeze-dried yolk antibody minimum dose treatment for duck viral hepatitis and results thereof

Therefore, the bivalent egg yolk antibody can play a good role in treating the type I and type III duck viral hepatitis viruses and can be clinically used.

It will be appreciated by those skilled in the art that the invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The embodiments disclosed above are therefore to be considered in all respects as illustrative and not restrictive. All changes which come within the scope of or equivalence to the invention are intended to be embraced therein.