CN112870347A - Method for developing porcine circovirus 2d type and porcine infectious pleuropneumonia combined inactivated vaccine - Google Patents

Abstract

The application relates to a porcine circovirus 2d type and porcine infectious pleuropneumonia bivalent inactivated vaccine and a preparation method thereof, the bivalent inactivated vaccine contains inactivated porcine circovirus 2d type and inactivated porcine infectious pleuropneumonia actinobacillus, and the porcine circovirus 2d type content in the bivalent inactivated vaccine is more than or equal to 10^6.0 TCID ₅₀The content of actinobacillus pleuropneumoniae in the first part of the pig is more than or equal to 6 multiplied by 10⁹ CFU/ml. The bivalent inactivated vaccine reduces the antigen-antigen interactionThe mutual interference is realized, the effect of respectively immunizing single seedlings can be achieved only by one-time immunization, the complex procedures of porcine circovirus type 2d and porcine contagious pleuropneumonia single seedling immunization are saved, and the time consumption and the labor consumption are low; and the dosage of the oily adjuvant is reduced, so that the cost is reduced, and the stress response of animals is also reduced.

Description

Method for developing porcine circovirus 2d type and porcine infectious pleuropneumonia combined inactivated vaccine

Technical Field

The application belongs to the field of veterinary vaccines, and particularly relates to a method for developing a porcine circovirus type 2d and porcine infectious pleuropneumonia bivalent inactivated vaccine.

Background

The porcine contagious pleuropneumonia is an important infectious disease of a respiratory system caused by actinobacillus pleuropneumoniae, mainly causes cellulosic, hemorrhagic and necrotic pneumonia accompanied with pleurisy of pigs, and rapidly dies in the most acute or acute disease course, and the morbidity and the mortality are more than 50 percent. The porcine circovirus is the smallest animal virus discovered so far, the mortality rate is different from 10% to 30%, and the death and culling rate of a serious pig farm can reach 40% when the disease is outbreaked. In intensive pig farms, both piglets infected with contagious pleuropneumonia and porcine circovirus cause significant economic losses.

Disclosure of Invention

The invention aims to provide a porcine circovirus type 2d and porcine contagious pleuropneumonia bivalent inactivated vaccine and a preparation method thereof, which comprises at least one porcine circovirus type 2d antigen and at least one inactivated porcine contagious pleuropneumonia actinobacillus antigen so as to prevent and treat diseases of porcine circovirus disease and porcine pleuropneumonia disease.

The technical scheme adopted by the invention is as follows: a porcine circovirus type 2d and porcine infectious pleuropneumonia bivalent inactivated vaccine comprises inactivated porcine circovirus type 2d, inactivated porcine infectious actinobacillus pleuropneumoniae and an adjuvant; the porcine circovirus type 2 content is more than or equal to 10^6.0 TCID ₅₀The content of the porcine infectious actinobacillus pleuropneumoniae is more than or equal to6×10⁹ CFU/ml。

Further, the porcine circovirus type 2d adjuvant is a sterile Carbopol aqueous solution with the concentration of 3%.

Further, the porcine infectious pleuropneumonia virus adjuvant is an oil-in-water adjuvant, the oil-in-water adjuvant takes Span85 and squalene as oil phases, and takes sterile PBS and Tween80 as water phases; the proportion of each component is Span 851%, squalene 5%, Tween 801%, and pH7.5 PBS.

Further, the volume ratio of the antigen to the adjuvant is 1: 1.

the invention adopts another technical scheme that: a preparation method of a porcine circovirus 2d type and porcine infectious pleuropneumonia combined inactivated vaccine comprises the following steps:

step 101, respectively preparing production seeds of porcine circovirus type 2d and porcine contagious pleuropneumonia virus;

103, preparing virus liquid for preparing the vaccine of porcine circovirus type 2d and porcine contagious pleuropneumonia respectively;

step 105, respectively adding 10% formaldehyde solution into virus liquid for preparing the vaccine of porcine circovirus type 2d and porcine contagious pleuropneumonia to inactivate, so that the final concentration of the formaldehyde solution is 0.2% (V/V), fully shaking up, then putting the mixture in an environment of 37 ℃ to inactivate for 24 hours, stirring the mixture for 3-5 times, and adding 0.2% sodium metabisulfite to terminate inactivation;

step 107, respectively carrying out 10-time concentration on virus liquid for preparing the vaccine of porcine circovirus type 2d and porcine contagious pleuropneumonia;

step 109, preparing porcine circovirus type 2d and porcine infectious pleuropneumonia adjuvants respectively, wherein the porcine circovirus type 2 adjuvant is a sterile Carbopol aqueous solution, and the porcine infectious pleuropneumonia viral adjuvant is an oil-in-water adjuvant;

and step 111, mixing the virus liquid for preparing the vaccine obtained in the step 107 with a corresponding virus adjuvant according to the volume ratio of 1:1, adding a diluent to dilute the mixture to the concentration before concentration, mixing the two virus liquids according to the volume ratio of 1:1, and filling the mixture into a sterile container to stir at 37 ℃ for 35 min.

Further, the diluent was sterile PBS buffer solution, pH 7.5.

The invention has the beneficial effects that: reducing mutual interference among antigens to obtain a porcine circovirus type 2d and infectious pleuropneumonia combined inactivated vaccine with good immunogenicity; the effect of respectively immunizing single seedlings can be achieved only by one-time immunization, the complex procedures of single seedling immunization of porcine circovirus type 2 and porcine infectious pleuropneumonia are saved, and the time consumption and the labor consumption are low; and the dosage of the oily adjuvant is reduced, so that the cost is reduced, and the stress response of animals is also reduced.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and it is obvious for those skilled in the art that other drawings can be obtained according to these drawings without creative efforts.

FIG. 1 is a schematic flow chart of an embodiment of the present invention.

Detailed Description

In order that the above objects, features and advantages of the present invention can be more clearly understood, a more particular description of the invention will be rendered by reference to the appended drawings. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, however, the present invention may be practiced in other ways than those specifically described herein, and thus the present invention is not limited to the specific embodiments disclosed below.

1. Preparation of production seeds

(1) Preparation of porcine circovirus type 2d production seeds

The virus seeds are properly diluted by sterile PBS, inoculated to PK15 cells for culture according to 5 percent, the PK15 cells are purchased from Chinese veterinary drug inspection departments, adsorbed for 45 minutes at 37 ℃, added with MEM cell maintenance liquid containing 5 percent of calf serum and 3mmol/L D-glucosamine hydrochloride, cultured for 5 days at 37 ℃, frozen and thawed for 4-5 times during the period, and then the virus is harvested.

(2) Preparation of seed for producing porcine infectious pleuropneumonia

The freeze-dried strains of the porcine infectious actinobacillus pleuropneumoniae are respectively streaked and inoculated on a TSA/NAD plate, the TSA/NAD plate contains 5 percent calf serum, and the plate is placed at 37 ℃ for culturing for 24 hours. And selecting a colony meeting the requirement, inoculating the colony in a TSB/NAD (containing 5% calf serum) liquid culture medium, and culturing at 37 ℃ for 12-16 hours to serve as a primary seed. Taking a culture of the primary seeds, adding the culture into a TSB/NAD (containing 5% calf serum) liquid culture medium according to the amount of 0.1%, culturing for 12-16 hours at 37 ℃, and checking to be pure to serve as secondary seeds.

2. Preparation of virus liquid

(1) Preparation of porcine circovirus type 2d virus liquid

Removing the culture medium from the monolayer of PK15 cells (purchased from ATCC), and mixing the seed solution with the culture medium at a temperature of 0.1-0.2 TCID₅₀The inoculation amount of each cell is inoculated on PK15 cells, the cell bottle is gently rotated for 2 weeks, the cells are adsorbed for 40 minutes at 37 ℃, cell maintenance liquid is added, and the cells are placed at 37 ℃ for rotating (12-16 r/h) for culture. Observing for 2-3 times every day, wherein the cell growth is good, culturing at 37 ℃ for 5 days to harvest cells and cell sap, freezing and thawing for 3 times, and storing at the temperature below-20 ℃ for no more than 2 months. Filtering the porcine circovirus type 2 virus liquid by using a hollow fiber filter column to remove cell fragments.

(2) Preparation of porcine infectious pleuropneumonia bacterial liquid

Adding 0.01-0.05% NAD and 5-10% serum into a porcine infectious pleuropneumonia actinobacillus culture medium (commercial TSB), adding the porcine infectious pleuropneumonia actinobacillus bacterial liquid into the culture medium according to the amount of 0.2% for culture, uniformly mixing, culturing for 15-20 hours at 37 ℃, and stopping culturing when the concentration OD600 of the bacterial liquid reaches more than 2.5, the DO value begins to rise and the PH value is reduced to be less than 6.5.

3. Inactivating

Adding 10% formaldehyde solution into porcine circovirus 2d type virus solution and porcine infectious pleuropneumonia virus solution for inactivation to ensure that the final concentration of the formaldehyde solution is 0.2% (V/V), fully shaking up, then putting the mixture in an environment of 37 ℃ for inactivation for 24 hours, stirring the mixture for 3-5 times, and adding 0.2% sodium metabisulfite to terminate inactivation.

4. Concentrating

(1) Concentration of porcine circovirus type 2d virus liquid

Concentrating porcine circovirus type 2d virus solution 10 times with Millipore membrane (molecular cut-off of 300Kda Dalton), and concentrating to obtain 10-fold concentrate^7.0 TCID₅₀ First part.

(2) Concentration of porcine infectious pleuropneumonia bacterial liquid

Using a hollow fiber ultrafilter (GE healthcare, UniFluxTM D, 30D-5 m)²) Concentrating by 10 times to obtain concentrate with content of 6 × 10¹⁰ CFU/ml。

5. Preparation of adjuvants

(1) Preparation of porcine circovirus type 2d adjuvant

A sterile aqueous solution of Carbopol was used at a concentration of 2%.

(2) Preparation of porcine infectious pleuropneumonia adjuvant

The porcine contagious pleuropneumonia adjuvant is an oil-in-water adjuvant, Span85 and squalene are used as oil phases, and sterile PBS and Tween80 are used as water phases; the proportion of each component is Span 851%, squalene 5%, Tween 801%, and pH7.5 PBS. Mixing sterile PBS (pH7.5) and Tween80 to obtain water phase, mixing Span85 and squalene to obtain oil phase, and stirring. Slowly adding the oil phase at 5000rpm, mixing the water phase and the oil phase, starting the emulsifying machine, and emulsifying for 15 min. The emulsified crude mixture was then cycled through the homogenizer 5 times, filtered and stored at 4 ℃ until use.

6. Preparing seedlings

Mixing virus solution for preparing vaccine with corresponding virus adjuvant at volume ratio of 1:1, adding diluent (sterile PBS buffer solution, pH 7.5) to dilute until the content of 2d type porcine circovirus is 1 × 10^6.0 TCID₅₀Per head, 6X 10 Actinobacillus pleuropneumoniae content⁹ CFU/ml. Mixing the two obtained virus solutions according to the volume ratio of 1:1, and placing the mixture into a sterile container to stir for 35min at the conditions of 37 ℃ and 3000-.

7. Examination of

Selecting 70 weaned piglets of 20-30 days old, and dividing into 7 groups, wherein each group has 10 piglets; 1ml of porcine circovirus type 2d and porcine infectious pleuropneumonia bivalent inactivated vaccine is injected into the neck muscle of each pig in the 1 st and 2 nd groups; in the 3 rd group, 1ml of porcine circovirus 2d type inactivated vaccine is injected into the neck of each pig through muscle, and 1ml is inoculated again after 14d of immunization; 4, injecting porcine infectious pleuropneumonia inactivated vaccine into neck muscle of each pig in the group 4; group 5 neck intramuscular injection of sterile PBS 1ml, 14d another inoculation of 1ml PBS; group 6 cervical muscle injection of sterile PBS 1 ml; group 7 was a blank control, neither inoculated nor challenged, and was raised in a separate room.

Test results show that the bivalent inactivated vaccine trial-prepared in the embodiment of the invention has the immune effect equivalent to the sum of single commercial vaccines in the market from the aspect of virus attack protection effect or serological antibody level, and the two antigens have no mutual interference; secondly, the bivalent inactivated vaccine has longer immune duration and lasting efficacy, and has immune protection on porcine contagious pleuropneumonia for at least 180 days and the porcine circovirus type 2d for at least 120 days; finally, the bivalent inactivated vaccine provided by the embodiment of the invention can achieve the effect of respectively immunizing single seedlings only by one-time immunization, and the complex procedure of 2d type single vaccine immunization of the porcine circovirus is omitted, so that the time consumption and the labor consumption are low, the cost is reduced, and the stress reaction of animals is also reduced.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (6)

1. A porcine circovirus type 2d and porcine infectious pleuropneumonia combined inactivated vaccine is characterized by comprising inactivated porcine circovirus type 2d, inactivated porcine infectious actinobacillus pleuropneumoniae and an adjuvant; the porcine circovirus type 2 content is more than or equal to 10^6.0 TCID ₅₀The content of actinobacillus pleuropneumoniae in swine is not less than 6 multiplied by 10⁹ CFU/ml。

2. The porcine circovirus type 2d, porcine infectious pleuropneumonia bivalent inactivated vaccine according to claim 1, wherein the porcine circovirus type 2d adjuvant is a sterile Carbopol aqueous solution with a concentration of 3%.

3. The porcine circovirus type 2d and porcine infectious pleuropneumonia combined inactivated vaccine as claimed in claim 1, wherein the porcine infectious pleuropneumonia virus adjuvant is an oil-in-water adjuvant, the oil-in-water adjuvant uses Span85 and squalene as oil phase, and uses sterile PBS and Tween80 as water phase; the proportion of each component is Span 851%, squalene 5%, Tween 801%, and pH7.5 PBS.

4. The porcine circovirus type 2 and porcine contagious pleuropneumonia combined inactivated vaccine according to claim 1, wherein the volume ratio of the antigen to the adjuvant is 1: 1.

5. a preparation method of a porcine circovirus 2d type and porcine infectious pleuropneumonia combined inactivated vaccine is characterized by comprising the following steps:

step 101, respectively preparing production seeds of porcine circovirus type 2d and porcine contagious pleuropneumonia;

103, preparing virus liquid for preparing the vaccine of porcine circovirus type 2d and porcine contagious pleuropneumonia respectively;

step 105, respectively adding 10% formaldehyde solution into virus liquid for preparing the vaccine of porcine circovirus type 2d and porcine contagious pleuropneumonia to inactivate, so that the final concentration of the formaldehyde solution is 0.2% (V/V), fully shaking up, then putting the mixture in an environment of 37 ℃ to inactivate for 24 hours, stirring the mixture for 3-5 times, and adding 0.2% sodium metabisulfite to terminate inactivation;

step 107, respectively carrying out 10-time concentration on virus liquid for preparing the vaccine of porcine circovirus type 2d and porcine contagious pleuropneumonia;

step 109, preparing porcine circovirus type 2d and porcine infectious pleuropneumonia adjuvants respectively, wherein the porcine circovirus type 2d adjuvant is a sterile Carbopol aqueous solution, and the porcine infectious pleuropneumonia adjuvant is an oil-in-water adjuvant;

and step 111, mixing the virus liquid for preparing the vaccine obtained in the step 107 with a corresponding virus adjuvant according to the volume ratio of 1:1, adding a diluent to dilute the mixture to the concentration before concentration, mixing the two virus liquids according to the volume ratio of 1:1, and filling the mixture into a sterile container to stir at 37 ℃ for 35 min.

6. The method for preparing the porcine circovirus type 2d and porcine infectious pleuropneumonia bivalent inactivated vaccine according to claim 5, wherein the diluent is a sterile PBS buffer solution and the pH value is 7.5.

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