KR100769356B1 - Erysipelothrix rhusiopathiae culture medium without animal serum and swine erysipelas live and killed vaccine prepared with the bacteria cultured on that medium - Google Patents

Abstract

The present inventors tested the immunogenicity in the development of pig mononuclear bacteria in a medium developed to improve the development of pig mononuclear bacteria and inactivated vaccines made from pig mononuclear bacteria grown in this medium, and obtained the following results. .

Don alone viable cell count showed about 10-fold improvement in bacterial counts compared to cultured by adding 5-10% bovine serum to conventional Brain Heart Infusion (BHI) medium.

After incubation at 37 ° C. for 18-20 hours, the culture medium showed a very stable pH of 7.0.

The antibody-forming ability of the pig mononuclear inactivated vaccine prepared with pig mononuclear bacteria cultured in the above development medium was good.

Don alone culture medium, no animal serum

Description

Erysipelothrix rhusiopathiae culture medium without animal serum and swine erysipelas live and killed vaccine prepared with the bacteria cultured on that medium}

The present invention relates to a pig mononuclear medium that can dramatically increase the number of bacteria without the use of animal serum.

Toxin vaccines produced so far are cultured in medium made by adding calf serum to Brain Heart infusion ("BHI") medium, but the productivity is low due to the small number of cultured bacteria Production costs are very high because fetal bovine serum (5-10% of the amount of medium) is expensive. In addition, since various heterologous proteins (fetal bovine serum) are contained in the medium, the side effects caused by the heterologous proteins are constantly present when inoculating vaccines made therefrom. In addition, the pH of the cultured medium is rapidly lowered, which affects the viability of the viable bacteria and thus adversely affects the preservation of the viable bacteria.

In order to solve the above problems, the present invention develops a toxin bacterium medium that can stably maintain the pH of the medium while significantly increasing the number of the money toxin bacteria without using animal serum in the toxin bacterium medium. Came to complete. Since the technology developed in the present invention is expected to greatly contribute to improving the productivity, reducing the production cost, and improving the safety and stability of the P. monobacterium vaccine, the possibility of industrialization is very high.

The present invention is proteose peptone NO.3 5g, yeast extract (Yeast extract, Difco 0127-17) 5g, sodium phosphate (Na ₂ HPO ₄ ) 12g, potassium dihydrogen phosphate (KH ₂ PO ₄ ) 2.1 g, brain heart infusion (BHI) 37g, the base medium consisting of 1,000ml of distilled water was adjusted to PH 7.4-7.5, 15 pounds (Lb), 20 minutes autoclave, and then filter sterilization Cultured in adontococci medium and its medium prepared by adding an arginine solution 0.05%, glucose solution 0.04%, gelatin solution 0.04%, and Tween 80 0.1% The present invention relates to a pig mononuclear inactivated vaccine prepared by using a virulent bacteriophage and its medium.

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In the improved medium of the present invention, in addition to the animal serum, the cultured pig monosaccharides were cultured by filtration and sterilization of essential ingredients Brain Heart Infusion (BHI) and arginine, glucose, gelatin, and Tween 80 so as to maximize the development of pig monosaccharides. Increased productivity than the medium can increase the productivity, not only reduce the production cost by using expensive animal serum, but also reduce the possibility of side effects of the vaccine due to heterologous protein, and also in the improved medium of the present invention In order to ensure the safety of single bacteria, the basic medium is prepared by applying an appropriate amount of sodium phosphate (Na ₂ HPO ₄ ) and potassium dihydrogen phosphate (KH ₂ PO ₄ ) to maintain the neutral pH even after germ development. It can also act positively on the stability of the live bacterial vaccine.

Hereinafter, the present invention will be described in detail by way of examples.

Example 1.Preparation of Porcine Aureus Medium

Badge composition

Prothios peptone N0.3 5g

5g yeast extract

Sodium Phosphate 12g

Potassium Dihydrogen Phosphate 2.1g

Brain Heart Infusion 37g

1,000ml of distilled water

After the pH 7.4-7.5 adjustment, add 15 pounds, 20 min wet heat sterilization and filter sterilization.

Arginine solution 0.05%

Glucose solution 0.04%

Gelatin solution 0.04%

Tween 80 0.1%

Experimental Example 1. Viable cell count and medium PH

As the strain used for the culture test, T2 strain, which is a strong poison strain, used as a pig-inactivated vaccine strain, was used. As a test medium, a medium in which 5% fetal bovine serum was added to the Brain Heart Infusion (BHI) medium, a medium without the Brain Heart Infusion component in the development medium, and three development mediums were used for the comparative test. Inoculation was carried out to 0.1% of the amount of the toxin bacteria by medium and incubated for 18-20 hours at 37 ℃. This was immediately tested for viable cells by standard plate culture method using a medium containing 10% immobilized calf serum to brain heart infusion agar and pH was measured using a pH meter (Mettler, MP220). Brain Heart Infusion Agar plates tested for viable cell number were cultured at 37 ° C. for 48 hours, and the number of living cells was examined with colony formed. Brain Heart Infusion medium was added to the brain heart infusion medium with 5% fetal bovine serum. The number of viable mononuclear cells grown in development medium was 52 X 10 ⁷ CFU / ml, 98 X 10 ⁷ CFU / ml, and 517 X 10 ⁷ CFU / ml, respectively. It was about 10 times more than cultured in brain heart infusion medium. In addition, the pH of the medium after 18 hours of cultivation was 7.0, which was more stable than that of 5.46 of medium cultured in the Brain Heart Infusion medium.

Table 1. Viable cell counts and pH of cultured P. aeruginosa

Badge Medium pH Don alone viable count (X 10 ⁷ CFU / ml) Average    One*    5.46    56, 55, 46 52 X 10 ⁷    2**    7.21    88, 89, 117 98 X 10 ⁷    3 ***    7.0    528, 492, 532 517 X 10 ⁷

1: Brain Heart Infusion (BHI) + 5% Fetal Bovine Serum

2: No Brain Heart Infusion (BHI) Ingredients in Development Medium (Arginine Medium)

3: development medium

Experimental Example 2. Antibody-forming ability of mouse incubated vaccine of S. aureus

Formarin was added to the culture of S. aureus for each medium, and inactivated for 4-5 days at room temperature. After confirming the inactivation of the bacteria, the vaccine was mixed well by adding 20 mg / ml of aluminum hydroxide gel to 15% of the culture and allowed to stand in a refrigerator at 5 ° C. for 5-10 days to remove about 50% of the supernatant. The vaccine was prepared by removal. Inactivated vaccines were inoculated subcutaneously with 0.2ml of 20-25 grams of ICR mice, and the antibody titers of blood antibody 2 weeks after each inoculation were examined by the growth agglutination test (GAT) method. . Two weeks after the first inoculation, the antibody titer of the vaccine cultured in two mediums, such as Brain Heart Infusion, was <4, which was lower than that of the development medium, which was lower than that of the development medium. Compared with 256 times, the immunogenicity of vaccines made from antigens cultured in conventional media was 8 times and 16 times lower in proportion to the number of bacteria (Table 2).

Table 2. Antibody Formation Ability in Mice of Don Dog Inactivated Vaccine Cultured in Development Medium

Belly stars Fungus content Antibody ^Value (GAT) ^* 2 weeks after the first vaccination 2 weeks after the 2nd vaccination  1.Brain Heart Infusion Broth + 5% Serum 52 X 10 ⁷ CFU / ml <4 8  2.Arginine Medium 98 X 10 ⁷ CFU / ml <4 16  3. Development medium 517 X 10 ⁷ CFU / ml 8 256

* Growth agglutination test (Growth agglutination test, GAT)

As described above, in the improved medium of the present invention, instead of animal serum, essential ingredients are filtered and sterilized so as to maximize the development of the toxin bacterium (arginine, glucose, gelatin, Tween 80) and brain heart infusion broth (BHI). By adding), the number of cultured pig mononuclear bacteria can be increased by more than 10 times compared to the conventional medium, which can increase productivity, and it can reduce the production cost by not using expensive animal serum, and also can cause side effects of the vaccine due to heterologous protein. In addition, in the improved medium of the present invention, an appropriate amount of sodium phosphate (Na ₂ HPO ₄ ) and potassium dihydrogen phosphate (KH) to maintain a neutral pH even after the growth of the bacterium in order to ensure the safety of pig mononuclear bacteria ₂ PO ₄ ) by applying a basic medium can be a positive effect on the stability of the virulent vaccine of S. aureus.

Claims (6)

After adjusting the base medium composed of 5 g of prothios peptone NO.3, 5 g of yeast extract, 12 g of sodium phosphate, 2.1 g of potassium dihydrogen phosphate, 37 g of brain heart infusion, and 1,000 ml of distilled water to pH 7.4-7.5, After 20 minutes of moist heat sterilization, the culture of Dontococcus medium prepared by adding 0.05% arginine solution, 0.04% glucose solution, 0.04% gelatin solution, and 0.1% Tween 80. The virulent vaccine of swine pneumoniae prepared from the swine monosaccharide cultured in the medium of claim 1. Toxin bacterium inactivated vaccine prepared by inactivating toxin cultured in the medium of claim 1. delete delete delete