阅读说明：本技术 一种呼肠孤病毒和细小病毒卵黄抗体锭及其制备方法 (Reovirus and parvovirus egg yolk antibody lozenge and preparation method thereof ) 是由 王锐 于 2019-10-09 设计创作，主要内容包括：本发明提供了一种呼肠孤病毒和细小病毒卵黄抗体锭及其制备方法,属于卵黄抗体锭领域。本发明采用锭制剂提取的新型呼肠孤病毒和细小病毒卵黄抗体提取率高,依次通过稀释、过滤、沉淀脂类、沉淀抗体、制软材包被、压锭的步骤,制备的卵黄抗体的纯度高,提取效率好,本发明制备的卵黄抗体在预防和治疗畜禽疾病方面具有特异性强、作用迅速、无残留等优点,尤其是在治疗与预防水禽呼肠孤病毒、细小病毒具有极佳疗效,而且,本发明的方法可以得到了广泛应用。本发明的方法来源广泛、成本低、无过敏反应且有营养作用,高免卵黄抗体锭,易于保存、运输和应用,可以用于制成各种畜禽疾病,尤其是水禽的呼肠孤病毒、细小病毒的治疗药物。(The invention provides a reovirus and parvovirus egg yolk immunoglobulin spindle and a preparation method thereof, belonging to the field of egg yolk immunoglobulin spindles. The novel reovirus and parvovirus egg yolk antibody extracted by the pastille preparation has high extraction rate, and the egg yolk antibody prepared by the method sequentially comprises the steps of diluting, filtering, precipitating lipids, precipitating antibodies, preparing soft material coating and pressing pastille, has high purity and good extraction efficiency, has the advantages of strong specificity, quick action, no residue and the like in the aspect of preventing and treating livestock and poultry diseases, particularly has excellent curative effect in treating and preventing the waterfowl reovirus and the parvovirus, and can be widely applied. The method has the advantages of wide source, low cost, no anaphylactic reaction, nutritional effect, high immunity of the yolk antibody lozenge, easy storage, transportation and application, and can be used for preparing medicaments for treating various livestock and poultry diseases, in particular reoviruses and parvoviruses of waterfowls.)

1. A preparation method of reovirus and parvovirus egg yolk immunoglobulin tablets is characterized by comprising the following steps:

(1) raising the immune chicken: selecting healthy 25-week-old chicks, carrying out isolated feeding, carrying out prime immunization by intramuscular injection of reovirus and parvovirus inactivated vaccines under chicken wings of the chicks, and then respectively carrying out enhanced immunization, and collecting eggs when the antibody level in serum of the chicks reaches 10log 2-14 log2 after the prime immunization is detected;

(2) and (3) disinfecting and refrigerating eggs: sequentially cleaning, disinfecting and airing the eggs, and then refrigerating at 4 ℃;

(3) egg yolk collection: beating the eggs refrigerated in the step (2), separating egg white from yolk at 4 ℃ under aseptic conditions, taking the yolk, and uniformly stirring the yolk;

(4) diluting yolk liquid: mixing the yolk obtained in the step (3) with water, and standing in a refrigerating chamber at 4 ℃ to obtain diluted yolk liquid;

(5) and (3) filtering: filtering the yolk diluent in the step (4) to obtain filtrate;

(6) precipitation of lipid material: mixing the filtrate obtained in the step (5), normal saline and carboxymethyl cellulose for precipitation to obtain supernatant;

(7) and (3) filtering: filtering the supernatant obtained in the step (6) to obtain filtrate;

(8) precipitated antibody material: mixing the filtrate obtained in the step (7) with sodium sulfate for precipitation, and taking solid substances;

(9) preparing ingots: and (3) mixing the solid substances obtained in the step (8), auxiliary materials, cane sugar and beta-cyclodextrin to obtain a soft material, and then sequentially granulating, drying and pressing into ingots to obtain the reovirus and parvovirus egg yolk antibody ingots.

2. The preparation method according to claim 1, wherein the volume ratio of the yolk to the water in the step (4) is 1: 2-4.

3. The preparation method according to claim 1 or 2, wherein the step (4) further comprises sterilization after mixing, and the sterilization comprises the following steps: and heating the mixed solution to 65-68 ℃, keeping the temperature for 300 seconds, and then cooling the mixed solution to 4-5 ℃ within 10-30 seconds.

4. The preparation method according to claim 1, wherein the volume ratio of the filtrate to the normal saline in the step (6) is 1: 2-4, and the obtained carboxymethyl cellulose is added in the form of a carboxymethyl cellulose solution, and the concentration of the carboxymethyl cellulose solution is 10-30 g/L.

5. The method according to claim 4, wherein the amount of the carboxymethyl cellulose solution used in the step (6) is 0.3 times the volume of the filtrate.

6. The method according to claim 1, wherein the precipitation in the steps (6) and (8) is carried out at a temperature of 10 to 15 ℃ for 12 to 24 hours.

7. The preparation method according to claim 1, wherein the sodium sulfate in the step (8) is added in the form of a sodium sulfate solution, and the concentration of the sodium sulfate solution is 10-30 g/L.

8. The preparation method according to claim 1, wherein the mass ratio of the solid matter to the auxiliary material in the step (9) is 1: 3-1: 6.

9. the preparation method according to claim 1, wherein the sucrose is used in an amount of 15 to 40% by mass of the solid substance, and the β -cyclodextrin is used in an amount of 1 to 3% by mass of the solid substance.

10. Reovirus and parvovirus yolk immunoglobulin produced by the production method according to any one of claims 1 to 9.

Technical Field

The invention relates to the technical field of yolk immunoglobulin tablets, in particular to a reovirus and parvovirus yolk immunoglobulin tablet and a preparation method thereof.

Background

The novel reovirus and parvovirus of waterfowls have high morbidity and mortality, thereby not only bringing great harm to the breeding industry, but also endangering the health of human beings. Reovirus has a plurality of serotypes, and is easy to generate drug resistance, thereby bringing difficulty to the treatment of diseases. Particularly, the disease is easy to cause in modern large-scale and intensive culture districts, and is often mixed with duck liver virus, avian influenza and the like to cause mass death, thus bringing huge economic loss to culture production. Inactivated vaccines are mainly adopted in China at present, but the waterfowl reovirus and the parvovirus have different using effects, the antibody protection time is short, and the waterfowl reovirus and the parvovirus can obtain stronger immunity after being immunized for multiple times. The waterfowl reovirus and parvovirus are quickly infected and have high pathogenicity, no composite vaccine with obvious immune protection is available at present, the inactivated vaccine has slow effect, the composite antibody effectively supplements the blank period of immunity, and the effect is quick and obvious. Therefore, antibodies of reoviruses and parvoviruses of waterfowls belong to the blank.

The yolk antibody has the advantages of strong specificity, quick action, no residue and the like in the aspect of preventing and treating livestock and poultry diseases, and is widely applied. Moreover, from the perspective of immunoprophylaxis and treatment, the hyperimmune egg yolk has the efficacy, wide sources, low cost, no anaphylaxis and nutritional effect. The high-immunity yolk antibody tablet is easy to store, transport and apply, and has been initially successful in clinical tests. Therefore, how to extract and preserve the effective titer of the yolk antibody is very important for the application of the yolk. The existing extraction method is a simple acidified water method, the separation rate is low, and the extraction titer is unstable.

Disclosure of Invention

In view of the above, the present invention aims to provide a reovirus and parvovirus yolk immunoglobulin tablet and a preparation method thereof. The reovirus and parvovirus egg yolk antibody lozenge prepared by the method can effectively ensure the titer of the antibody and can improve the absorption utilization rate of the antibody.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a preparation method of reovirus and parvovirus yolk immunoglobulin tablets, which comprises the following steps:

(1) raising the immune chicken: selecting healthy 25-week-old chicks, carrying out isolated feeding, carrying out prime immunization by intramuscular injection of reovirus and parvovirus inactivated vaccines under chicken wings of the chicks, and then respectively carrying out enhanced immunization, and collecting eggs when the antibody level in serum of the chicks reaches 10log 2-14 log2 after the prime immunization is detected;

(2) and (3) disinfecting and refrigerating eggs: sequentially cleaning, disinfecting and airing the eggs, and then refrigerating at 4 ℃;

(3) egg yolk collection: beating the eggs refrigerated in the step (2), separating egg white from yolk at 4 ℃ under aseptic conditions, taking the yolk, and uniformly stirring the yolk;

(4) diluting yolk liquid: mixing the yolk obtained in the step (3) with water, and standing in a refrigerating chamber at 4 ℃ to obtain diluted yolk liquid;

(5) and (3) filtering: filtering the yolk diluent in the step (4) to obtain filtrate;

(6) precipitation of lipid material: mixing the filtrate obtained in the step (5), normal saline and carboxymethyl cellulose for precipitation to obtain supernatant;

(7) and (3) filtering: filtering the supernatant obtained in the step (6) to obtain filtrate;

(8) precipitated antibody material: mixing the filtrate obtained in the step (7) with sodium sulfate for precipitation, and taking solid substances;

(9) preparing ingots: and (3) mixing the solid substances obtained in the step (8), auxiliary materials, cane sugar and beta-cyclodextrin to obtain a soft material, and then sequentially granulating, drying and pressing into ingots to obtain the reovirus and parvovirus egg yolk antibody ingots.

Preferably, the volume ratio of the egg yolk to the water in the step (4) is 1: 2-4.

Preferably, the step (4) further comprises sterilization after mixing, and the sterilization process comprises: and heating the mixed solution to 65-68 ℃, keeping the temperature for 300 seconds, and then cooling the mixed solution to 4-5 ℃ within 10-30 seconds.

Preferably, the volume ratio of the filtrate to the normal saline in the step (6) is 1: 2-4, the obtained carboxymethyl cellulose is added in the form of carboxymethyl cellulose solution, and the concentration of the carboxymethyl cellulose solution is 10-30 g/L.

Preferably, the amount of the carboxymethyl cellulose solution used in the step (6) is 0.3 times of the volume of the filtrate.

Preferably, the temperature of the precipitation in the steps (6) and (8) is 10-15 ℃ independently, and the time is 12-24 h independently.

Preferably, the sodium sulfate in the step (8) is added in the form of a sodium sulfate solution, and the concentration of the sodium sulfate solution is 10-30 g/L.

Preferably, the mass ratio of the solid matters to the auxiliary materials in the step (9) is 1: 3-1: 6.

preferably, the amount of the sucrose is 15-40% of the mass of the solid matter, and the amount of the beta-cyclodextrin is 1-3% of the mass of the solid matter.

The invention also provides reovirus and parvovirus yolk immunoglobulin tablets prepared by the preparation method in the technical scheme.

The invention provides a preparation method of reovirus and parvovirus yolk immunoglobulin tablets, which comprises the following steps: (1) raising the immune chicken: selecting healthy 25-week-old chicks, carrying out isolated feeding, carrying out prime immunization by intramuscular injection of reovirus and parvovirus inactivated vaccines under chicken wings of the chicks, and then respectively carrying out enhanced immunization, and collecting eggs when the antibody level in serum of the chicks reaches 10log 2-14 log2 after the prime immunization is detected; (2) and (3) disinfecting and refrigerating eggs: sequentially cleaning, disinfecting and airing the eggs, and then refrigerating at 4 ℃; (3) egg yolk collection: beating the eggs refrigerated in the step (2), separating egg white from yolk at 4 ℃ under aseptic conditions, taking the yolk, and uniformly stirring the yolk; (4) diluting yolk liquid: mixing the yolk obtained in the step (3) with water, and standing in a refrigerating chamber at 4 ℃ to obtain diluted yolk liquid; (5) and (3) filtering: filtering the yolk diluent in the step (4) to obtain filtrate; (6) precipitation of lipid material: mixing the filtrate obtained in the step (5), normal saline and carboxymethyl cellulose for precipitation to obtain supernatant; (7) and (3) filtering: filtering the supernatant obtained in the step (6) to obtain filtrate; (8) precipitated antibody material: mixing the filtrate obtained in the step (7) with sodium sulfate for precipitation, and taking solid substances; (9) preparing ingots: and (3) mixing the solid substances obtained in the step (8), auxiliary materials, cane sugar and beta-cyclodextrin to obtain a soft material, and then sequentially granulating, drying and pressing into ingots to obtain the reovirus and parvovirus egg yolk antibody ingots.

Compared with the prior art, the invention has the beneficial effects that:

the novel reovirus and parvovirus egg yolk antibody extracted by the pastille preparation has high extraction rate, and the egg yolk antibody prepared by the method sequentially comprises the steps of diluting, filtering, precipitating lipids, precipitating antibodies, preparing soft material coating and pressing pastille, has high purity and good extraction efficiency, has the advantages of strong specificity, quick action, no residue and the like in the aspect of preventing and treating livestock and poultry diseases, particularly has excellent curative effect in treating and preventing the waterfowl reovirus and the parvovirus, and can be widely applied. The method has the advantages of wide source, low cost, no anaphylactic reaction, nutritional effect, high immunity of the yolk antibody lozenge, easy storage, transportation and application, and can be used for preparing medicaments for treating various livestock and poultry diseases, in particular reoviruses and parvoviruses of waterfowls.

Detailed Description

The invention provides a preparation method of reovirus and parvovirus yolk immunoglobulin tablets, which comprises the following steps:

(1) raising the immune chicken: selecting healthy 25-week-old chicks, carrying out isolated feeding, carrying out prime immunization by intramuscular injection of reovirus and parvovirus inactivated vaccines under chicken wings of the chicks, and then respectively carrying out enhanced immunization, and collecting eggs when the antibody level in serum of the chicks reaches 10log 2-14 log2 after the prime immunization is detected;

(2) and (3) disinfecting and refrigerating eggs: sequentially cleaning, disinfecting and airing the eggs, and then refrigerating at 4 ℃;

(3) egg yolk collection: beating the eggs refrigerated in the step (2), separating egg white from yolk at 4 ℃ under aseptic conditions, taking the yolk, and uniformly stirring the yolk;

(4) diluting yolk liquid: mixing the yolk obtained in the step (3) with water, and standing in a refrigerating chamber at 4 ℃ to obtain diluted yolk liquid;

(5) and (3) filtering: filtering the yolk diluent in the step (4) to obtain filtrate;

(6) precipitation of lipid material: mixing the filtrate obtained in the step (5), normal saline and carboxymethyl cellulose for precipitation to obtain supernatant;

(7) and (3) filtering: filtering the supernatant obtained in the step (6) to obtain filtrate;

(8) precipitated antibody material: mixing the filtrate obtained in the step (7) with sodium sulfate for precipitation, and taking solid substances;

(9) preparing ingots: and (3) mixing the solid substances obtained in the step (8), auxiliary materials, cane sugar and beta-cyclodextrin to obtain a soft material, and then sequentially granulating, drying and pressing into ingots to obtain the reovirus and parvovirus egg yolk antibody ingots.

The invention raises immune chicks: selecting healthy 25-week-old chicks, carrying out isolated feeding, carrying out prime immunization by intramuscular injection of reovirus and parvovirus inactivated vaccines under chicken wings of the chicks, and then respectively carrying out enhanced immunization, and collecting eggs when the antibody level in serum of the chicks reaches 10log 2-14 log2 after the prime immunization. In the present invention, the boosting of the reovirus is preferably performed one week after priming, and the boosting of the parvovirus is preferably performed two weeks after priming. In the present invention, the amount of the booster vaccine is preferably the same as that used for priming.

The invention carries out the disinfection and refrigeration of eggs: and (3) sequentially cleaning, disinfecting and airing the eggs, and then refrigerating at 4 ℃.

The invention collects yolk: and (3) beating the refrigerated eggs, separating egg white from yolk at 4 ℃ under aseptic conditions, taking the yolk, and uniformly stirring the yolk. In the invention, a stirrer is preferably used for uniformly stirring the egg yolk, the stirring speed of the stirrer is preferably 150-200 r/min, and the stirring time is preferably 15-20 min.

The diluted egg yolk liquid of the invention: mixing the yolk with water, and standing in a refrigerating chamber at 4 deg.C to obtain diluted yolk liquid. In the invention, the volume ratio of the egg yolk to water is preferably 1: 2-4.

In the present invention, the mixing preferably further comprises sterilization, and the sterilization process is preferably as follows: and heating the mixed solution to 65-68 ℃, keeping the temperature for 300 seconds, and then cooling the mixed solution to 4-5 ℃ within 10-30 seconds.

The yolk diluent is filtered, and filtrate is obtained. In the present invention, the filtration is preferably a filtration membrane. In the present invention, the filtration membrane preferably has a pore size of 15 μm.

The present invention precipitates lipid material: and mixing the filtrate, normal saline and carboxymethyl cellulose for precipitation to obtain supernatant. In the invention, the volume ratio of the filtrate to the normal saline is preferably 1: 2-4, the obtained carboxymethyl cellulose is preferably added in the form of carboxymethyl cellulose solution, the concentration of the carboxymethyl cellulose solution is preferably 10-30 g/L, and the dosage of the carboxymethyl cellulose solution is preferably 0.3 times of the volume of the filtrate.

In the invention, the temperature of the precipitation is preferably 10-15 ℃, and the time is preferably 12-24 h.

The invention filters the supernatant and takes the filtrate. In the present invention, the filtration is preferably a sieving filtration, the sieving filtration is preferably a hollow fiber filter, and the molecular weight of the sieved antibody of the hollow fiber filter is preferably 5 to 10 ten thousand.

The present invention precipitates antibody substances: and mixing the filtrate with sodium sulfate for precipitation, and taking solid substances. In the invention, the sodium sulfate is preferably added in the form of a sodium sulfate solution, and the concentration of the sodium sulfate solution is preferably 10-30 g/L.

In the invention, the temperature of the precipitation is preferably 10-15 ℃, and the time is preferably 12-24 h.

The invention is used for ingot making: and mixing the solid matter, the auxiliary materials, the sucrose and the beta-cyclodextrin to obtain a soft material, and then sequentially granulating, drying and pressing into ingots to obtain the reovirus and parvovirus egg yolk antibody ingots.

In the present invention, the mass ratio of the solid substance to the auxiliary material is preferably 1: 3-1: 6. the invention is not limited to the kind of the auxiliary materials, and the kinds known to those skilled in the art can be used.

In the invention, the dosage of the sucrose is preferably 15-40% of the mass of the solid matter, and the dosage of the beta-cyclodextrin is preferably 1-3% of the mass of the solid matter.

In the invention, the encapsulation rate of the beta-cyclodextrin and the sucrose on solid matters and auxiliary materials is preferably over 90 percent.

In the invention, the drying is preferably carried out in a hot air circulation dryer, the drying temperature is preferably 60-75 ℃, and the drying time is preferably 1-3 hours.

In the present invention, the pressing into an ingot is preferably performed by pressing 100 g per ingot using an ingot press, and then packaged in an aluminum foil bag, and refrigerated at 4 ℃.

In order to further illustrate the present invention, the reovirus and parvovirus yolk antibody conjugate and the method for producing the same according to the present invention will be described in detail with reference to examples, which should not be construed as limiting the scope of the present invention.