CN1323718C - Process for preparing viral and bacterial vaccine for animals utilizing vegetable oil adjuvant - Google Patents

Abstract

A method utilizing vegetable oil adjuvant to prepare animal viral and bacterial vaccines is characterized in that it comprises the following steps: (1) making an antigenic agent, replicating the seedling virus (bacteria) strain with sensitive cells or culture medium, inactivating Afterwards, the antigen is obtained; and the antigen is made into an antigen agent; (2) making a vegetable oil adjuvant; (3) mixing, the antigen agent is mixed with the vegetable oil adjuvant in proportion, homogeneous, Make a vaccine. The antigen agent is a mixture of antigen and sterilized Tween-80; the vegetable oil adjuvant is a mixture of vegetable oil and Span-80. The present invention adopts novel adjuvant to prepare animal viral and bacterial vaccines, so that when applied, it will not affect the immune efficacy of vaccines, but also reduce the adverse reactions of vaccines, and at the same time, it is beneficial to produce safe food meat; Compared with other technologies, it has obvious advantages. The invention is suitable for use in various animal viral and bacterial vaccines.

Description

A method for preparing animal viral and bacterial vaccines using vegetable oil adjuvant

Technical field:

The invention relates to a method for producing vaccines using vegetable oil adjuvants, in particular to a method for preparing animal viral and bacterial vaccines using vegetable oil adjuvants.

Background technique:

Adjuvant, as a substance that can assist antigen to enhance immune response, can help antigen to be released slowly at the injection site to increase the effect of immune stimulation. In the existing technology, mineral oil adjuvants are more common in the application of animal vaccines, but in recent years, in the immune prevention of animal mineral oil adjuvant vaccines, due to frequent immune reactions, some of the animals injected with vaccines High fever, food reduction, food waste, miscarriage, and decline in production performance, such as reduced milk production of dairy animals, slowed weight gain of young animals and other adverse reactions, especially the death of a very small number of animals due to allergic reactions, are not conducive to the development of animal husbandry. develop. In addition, mineral oil (white oil) is a substance extracted from crude oil by chemical methods. When it is injected into animals as a vaccine adjuvant, it will be slowly metabolized for a long time, and even a very small amount of components will remain locally, which will affect the quality of animal meat. Also affect human health. Therefore, it is an important topic before us to study a new type of adjuvant, so that it will not affect the immune efficacy of the vaccine, but also reduce the adverse reaction of the vaccine, and at the same time be conducive to the production of safe meat products.

content of the invention

The object of the present invention is to provide a kind of method that utilizes vegetable oil adjuvant to prepare animal virality, bacterial vaccine, to solve the above-mentioned existing problems, thereby obtain neither affecting the immune effect of vaccine, but also can reduce the adverse reaction of vaccine, and at the same time Animal vaccines for the production of safe meat products.

The purpose of the present invention is achieved according to the following technical solutions. The present invention utilizes vegetable oil adjuvant to prepare the method for animal virus, bacterial vaccine, it is characterized in that it comprises the following steps:

(1) Making an antigenic agent, copying the seedling virus (bacteria) strain with sensitive cells or medium, and inactivating it to obtain the antigen; and making the antigen into an antigenic agent; it can be prepared according to conventional methods in this industry.

(2) making vegetable oil adjuvant;

(3) Mixing, mixing the antigen agent with the vegetable oil adjuvant in proportion, homogenizing, and preparing a vaccine.

In step (1),

The vaccine-making virus (bacteria) strain is any one or two of the following: O-type foot-and-mouth disease virus Akesu/58 strain, OMIII strain, and OZ strain, rabies virus-CVS strain, infectious gastrointestinal Inflammation (TEGV-TS strain), epidemic diarrhea virus (PEDV-QD strain), subtype avian influenza strain H5N1, subtype avian influenza strain H9N2, Salmonella pullorum; in addition, other animal viral, Bacterial vaccines.

Described sensitive cell is any one in the following: BHK-21 cell line and IB-RS-2 cell, Vero cell, PK-cell, 293 cell, ST-cell, Hela-cell; Can also adopt other sensitive cells cell.

The medium is any one of the following: LB, sodium thiosulfate agar, common broth, common agar plate. Other media can also be used.

In step (1),

The antigenic agent is a mixture of antigen and sterilized Tween-80;

In step (2),

The vegetable oil adjuvant is a mixture of vegetable oil and Span-80.

The vegetable oil is any one of the following: rapeseed oil, soybean oil, peanut oil, castor oil, flax oil and other vegetable oils. Other edible oils that are harmless to humans and animals can also be used.

In the step (1), making the antigen into an antigenic agent is as follows: mixing the antigen with sterilized Tween-80 in a ratio of 10-40:1 to make an antigenic agent;

In the step (2), the preparation of the vegetable oil adjuvant is as follows: mixing the vegetable oil and Span-80 at a ratio of 5-15:1, and sterilizing to prepare the vegetable oil adjuvant;

In the step (3), the antigenic agent is mixed with the vegetable oil adjuvant in proportion as follows: the antigenic agent and the vegetable oil adjuvant are mixed in a ratio of 0.5-1.5:1, stirred, and formed Homogeneous colostrum; the homogenization is: high-speed shearing and emulsification homogenization of the colostrum with a high-pressure homogenizer.

In the step (3), when the antigenic agent is mixed with the vegetable oil adjuvant, first add part of the antigenic agent to the whole vegetable oil adjuvant, and stir while adding to form uniform colostrum; The rest of the antigenic agent is added to the homogeneous colostrum.

The present invention uses vegetable oil as the component of the adjuvant to prepare the vegetable oil adjuvant. Vegetable oil is a natural substance extracted from plant seeds. Injecting it into animals as an adjuvant will neither affect the quality of animal food nor affect the health of people who eat the animal product. At the same time, it has the properties of mineral oil adjuvant : ① It can increase the surface area of the antigen and be easily swallowed by macrophages; ② Prolong the retention period of the antigen in the body and increase the chance of contact with immune cells; ③ Induce the inflammatory response of the antigen injection site and its local lymph nodes, which is beneficial to Stimulates the proliferation of immune cells. Therefore, vegetable oil adjuvants must be the best substitutes for mineral oil adjuvants in the vaccine development process.

In summary, the present invention uses novel adjuvants to prepare animal viral and bacterial vaccines, so that when they are applied, they will not affect the immune efficacy of the vaccines, can reduce the adverse reactions of the vaccines, and are conducive to the production of safe food. Meat; Compared with the prior art, it has obvious advantages. The invention is suitable for use in various animal viral and bacterial vaccines.

Detailed ways

Example 1

This embodiment is the animal virus subtype avian influenza H5N1 and H9N2 vegetable oil adjuvant inactivated mixed vaccine prepared by utilizing vegetable oil adjuvant, and its preparation method comprises the following steps:

(1) making an antigenic agent, replicating the vaccine strain with sensitive cells or culture medium, after inactivation, obtaining the antigen and making the antigen into an antigenic agent; wherein:

1) Virus strains: the popular strains H5N1 and H9N2 (1:1 ratio) for seedling production, the biological characteristics of the strains for seedling production: half infection dose (EID ₅₀ ) of chicken embryos is 8.25log2/ml, that is, seed virus The virus content is that every ml contains 8.25 EID ₅₀ ; the average death time (MDT) of chicken embryos is 72 hours after inoculation of 0.1ml in the allantoic cavity; The HA of 1% SPF chicken red blood cells is more than 8log2; the degree of purity is free from any foreign virus and bacterial contamination.

2) Replication and inactivation of virus: inoculate 0.1ml of SPF chicken embryo 9-11 days old in the allantoic cavity, detect the HA titer of chicken embryo allantoic fluid by 72-hour HA test, and harvest chicken embryo allantoic fluid with HA≥8log2 , add formaldehyde solution, inactivate at 37°C to form antigen.

The inactivated antigen was inoculated into an appropriate culture medium (ordinary agar plate), and the bacteria, mold and mycoplasma were tested. 0.1 ml of the inactivated allantoic fluid was inoculated with 9-11-day-old chicken embryos through the allantoic cavity, and blindly passed for 3 consecutive days. generation.

3) Mix the inactivated allantoic fluid with autoclaved Tween-80 at a ratio of 25:1 to make an antigenic agent,

(2) making vegetable oil adjuvant;

Mix and emulsify vegetable oil (soybean oil) and Span-80 at a ratio of 10:1, and then sterilize under high pressure at 1.5Mpa for 30 minutes to make a vegetable oil adjuvant;

(3) The said antigenic agent is mixed with the said vegetable oil adjuvant in proportion, homogeneously prepared into a vaccine. Mix the antigenic agent with the vegetable oil adjuvant at a ratio of 1:1. In this embodiment, first add 40-60% of the inactivated antigenic agent to the vegetable oil adjuvant, and stir while adding to form a Uniform colostrum, then on the basis of making a water-in-oil emulsion, add the rest of the antigen agent to the vegetable oil adjuvant for further emulsification. Then it is sheared and emulsified at a high speed by a high-pressure homogenizer to make a milky white vaccine.

Example 2

Present embodiment is the porcine type O foot-and-mouth disease inactivated vaccine that utilizes vegetable oil adjuvant to prepare, and its preparation method comprises the following steps:

(1) making an antigenic agent, replicating the vaccine strain with sensitive cells or culture medium, after inactivation, obtaining the antigen and making the antigen into an antigenic agent; wherein:

1) Virus strain: The vaccine strain is porcine O-type foot-and-mouth disease virus OZ strain

The biological characteristics of the seedling strain: the seedling strain should conform to the characteristics of O-type foot-and-mouth disease virus. Suckling rat poison is poisoned with O-type foot-and-mouth disease porcine blister skin poison through 6-8 day-old suckling mice, 4-5 generations of suckling mouse muscle poison, diluted with phosphate buffer solution of PH7.6-7.8, and subcutaneously injected on the back of the neck for 3 - 0.2ml/mouse for 4-day-old suckling rats, each titer should be no less than 4, and the LD50 should not be less than 8.5 after observing for 5 days.

Rejuvenation of virus seeds for seedling production: cytotoxic seeds should be rejuvenated for no less than 3 generations, which should meet the standards. The number of consecutive production generations shall not exceed 14 generations. The degree of purity is free from any foreign virus and bacterial contamination.

2) Replication and inactivation of the virus: use 3-14 generations of seedling cytotoxic seeds to inoculate the BHK-21 cell line and IB-RS-2 cells that have formed a good monolayer, and collect the cytovenom. Inactivate those that meet the standard through sterility test and toxicity determination. Add a certain amount of inactivator BEI to the venom that removes cell fragments, raise the temperature to 30°C, and inactivate for 28 hours. Store at 5°C.

The inactivated antigen was inoculated into the broth, and tested for bacteria, mold and mycoplasma. The inactivated antigen was subcutaneously injected into the neck of 4-5 3-4-day-old suckling mice, 0.2ml/only, and 2 healthy blank control mice were set up, observed for 7 days, and all suckling mice were free from foot-and-mouth disease symptoms and death .

3) Mix the inactivated antigen with high-pressure sterilized Tween-80 at a ratio of 25:1 to prepare an antigen agent.

(2) making vegetable oil adjuvant;

Mix and emulsify vegetable oil (soybean oil) and Span-80 at a ratio of 10:1, and then sterilize under high pressure at 1.5Mpa for 30 minutes to make a vegetable oil adjuvant;

(3) The said antigenic agent is mixed with the said vegetable oil adjuvant in proportion, homogeneously prepared into a vaccine.

Mix the antigenic agent with the vegetable oil adjuvant at a ratio of 1:1. In this embodiment, first add 40-60% of the inactivated antigenic agent to the vegetable oil adjuvant, and stir while adding to form a Uniform colostrum, then on the basis of making a water-in-oil emulsion, add the rest of the antigen agent to the vegetable oil adjuvant for further emulsification. Then it is sheared and emulsified at a high speed by a high-pressure homogenizer to make a milky white vaccine.

Example 3

Present embodiment is the bovine type O foot-and-mouth disease inactivated vaccine that utilizes vegetable oil adjuvant to prepare, and its preparation method comprises the following steps:

(1) making an antigenic agent, copying the vaccine-producing virus (bacteria) strain with sensitive cells or medium, after inactivation, obtaining the antigen and making the antigen into an antigenic agent; wherein:

1) Virus strain: The seedling strain is bovine O-type foot-and-mouth disease virus Akesu/58 strain Biological characteristics of the seedling strain: The seedling strain should conform to the characteristics of O-type foot-and-mouth disease virus. Virus seed is carried out 1: 1000 times dilutions with PBS (phosphate buffered saline), upper alcohol scratches and tongue surface smears 2-4 years old cattle 2ml without foot-and-mouth disease O type serum antibody, in 24-48 hours, test cattle Typical secondary lesions of FMD should appear.

Rejuvenation of virus seeds for seedling production: cytotoxic seeds should be rejuvenated for no less than 3 generations, which should meet the standards. The number of consecutive production generations shall not exceed 14 generations. The degree of purity is free from any foreign virus and bacterial contamination.

2) Replication and inactivation of the virus: use 3-14 generations of seedling cytotoxic seeds to inoculate the BHK-21 cell line and IB-RS-2 cells that have formed a good monolayer, and collect the cytovenom. Inactivate those that meet the standard through sterility test and toxicity determination. Add a certain amount of inactivator BEI to the venom that removes cell fragments, raise the temperature to 30°C, and inactivate for 28 hours. Store at 5°C.

The inactivated antigens were inoculated on ordinary agar plates, and tested for bacteria, molds and mycoplasma. The inactivated antigen was subcutaneously injected into the neck of 4-5 3-4-day-old suckling mice, 0.2ml/only, and 2 healthy blank control mice were set up, observed for 7 days, and all suckling mice were free from foot-and-mouth disease symptoms and death .

3) Mix the inactivated antigen with autoclaved Tween-80 at a ratio of 25:1 to make an antigen agent,

(2) making vegetable oil adjuvant;

Mix and emulsify vegetable oil (soybean oil) and Span-80 at a ratio of 10:1, and then sterilize under high pressure at 1.5Mpa for 30 minutes to make a vegetable oil adjuvant;

(3) The said antigenic agent is mixed with the said vegetable oil adjuvant in proportion, homogeneously prepared into a vaccine.

Mix the antigenic agent with the vegetable oil adjuvant at a ratio of 1:1. In this embodiment, first add 40-60% of the inactivated antigenic agent to the vegetable oil adjuvant, and stir while adding to form a Uniform colostrum, then on the basis of making a water-in-oil emulsion, add the rest of the antigen agent to the vegetable oil adjuvant for further emulsification. Then it is sheared and emulsified at a high speed by a high-pressure homogenizer to make a milky white vaccine.

Example 4

Present embodiment is the porcine type O foot-and-mouth disease inactivated vaccine (concentrated type) that utilizes vegetable oil adjuvant to prepare, and its preparation method comprises the following steps:

(1) making an antigenic agent, copying the vaccine-producing virus (bacteria) strain with sensitive cells or medium, after inactivation, obtaining the antigen and making the antigen into an antigenic agent; wherein:

1) Virus strain: The vaccine strain is porcine O-type foot-and-mouth disease virus OZ strain

The biological characteristics of the seedling strain: the seedling strain should conform to the characteristics of O-type foot-and-mouth disease virus. Suckling rat poison is poisoned with O-type foot-and-mouth disease porcine blister skin poison through 6-8 day-old suckling mice, 4-5 generations of suckling mouse muscle poison, diluted with phosphate buffer solution of PH7.6-7.8, and subcutaneously injected on the back of the neck for 3 - 0.2ml/mouse for 4-day-old suckling rats, each titer should be no less than 4, and the LD50 should not be less than 8.5 after observing for 5 days.

Rejuvenation of virus seeds for seedling production: cytotoxic seeds should be rejuvenated for no less than 3 generations, which should meet the standards. The number of consecutive production generations shall not exceed 14 generations. The degree of purity is free from any foreign virus and bacterial contamination.

2) Replication and inactivation of the virus: use 3-14 generations of seedling cytotoxic seeds to inoculate the BHK-21 cell line and IB-RS-2 cells that have formed a good monolayer, and the addition of the virus culture solution is based on each square Centimeter cell monolayer 0.1-0.3ml, collect cell venom. Inactivate those that meet the standard through sterility test and toxicity determination. Add a certain amount of inactivator BEI to the venom that removes cell fragments, raise the temperature to 30°C, and inactivate for 28 hours. Store at 5°C.

Inoculate the appropriate culture medium (broth) with the inactivated antigen, and carry out the inspection of bacteria, mold and mycoplasma. The inactivated antigen was subcutaneously injected into the neck of 4-5 3-4-day-old suckling mice, 0.2ml/only, and 2 healthy blank control mice were set up, observed for 7 days, and all suckling mice were free from foot-and-mouth disease symptoms and death .

3) Mix the inactivated antigen with high-pressure sterilized Tween-80 at a ratio of 25:1 to prepare an antigen agent.

(2) making vegetable oil adjuvant;

Mix and emulsify vegetable oil (peanut oil) and Span-80 at a ratio of 10:1, and then sterilize under high pressure at 1.5Mpa for 30 minutes to make a vegetable oil adjuvant;

(3) The said antigenic agent is mixed with the said vegetable oil adjuvant in proportion, homogeneously prepared into a vaccine. Mix the antigenic agent with the vegetable oil adjuvant at a ratio of 1:1. In this embodiment, first add 50% of the inactivated antigenic agent to the vegetable oil adjuvant, and stir while adding to form a uniform Colostrum, then on the basis of making a water-in-oil emulsion, add the rest of the antigen agent to the vegetable oil adjuvant for further emulsification. Then it is sheared and emulsified at a high speed by a high-pressure homogenizer to make a milky white vaccine.

Claims (1)

1, a kind of method utilizing vegetable oil adjuvant to prepare animal virus and/or bacterial vaccine is characterized in that it comprises the following steps: (1) making an antigenic agent, replicating the seedling strain and/or bacterial strain with sensitive cells or medium, and obtaining the antigen after inactivation; and making the antigen into an antigenic agent; (2) making vegetable oil adjuvant; (3) mixing, mixing the antigen agent with the vegetable oil adjuvant in proportion, homogenizing, and making a vaccine; In the step (1), the antigenic agent is a mixture of antigen and sterilized Tween-80; In the step (2), the vegetable oil adjuvant is a mixture of vegetable oil and Span-80; In the step (1), making the antigen into an antigenic agent is as follows: mixing the antigen with sterilized Tween-80 in a ratio of 10-40:1 to make an antigenic agent; In the step (2), the preparation of the vegetable oil adjuvant is as follows: mixing the vegetable oil and Span-80 at a ratio of 5-15:1, and sterilizing to prepare the vegetable oil adjuvant; In the step (3), the antigenic agent is mixed with the vegetable oil adjuvant in proportion as follows: the antigenic agent and the vegetable oil adjuvant are mixed in a ratio of 0.5-1.5:1, stirred, and formed Uniform colostrum; the homogenization is: high-speed shearing and emulsification homogenization of the colostrum with a high-pressure homogenizer; In the step (3), when the antigenic agent is mixed with the vegetable oil adjuvant, first add part of the antigenic agent to the whole vegetable oil adjuvant, and stir while adding to make it form a uniform the colostrum; then add the rest of the antigen agents to the uniform colostrum.