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A kind of preparation method of diphtheria vaccine

A diphtheria vaccine and diphtheria bacillus technology, applied in the field of vaccines, can solve the problems of long production cycle of diphtheria vaccine, excessive impurity protein in the final product, and large side reactions of the product, so as to reduce the risk of pollution, low impurity protein content, and high recovery rate. Effect

Active Publication Date: 2015-08-26
SHANDONG YIDU BIOTECH
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  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The invention proposes a preparation method of diphtheria vaccine, which solves the problems in the prior art that the production cycle of diphtheria vaccine is too long, ammonium sulfate pollution occurs in the production process, the final product has too much miscellaneous protein, and the product side reaction is too large

Method used

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  • A kind of preparation method of diphtheria vaccine
  • A kind of preparation method of diphtheria vaccine
  • A kind of preparation method of diphtheria vaccine

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preparation example Construction

[0022] like figure 1 Shown, a kind of preparation method of diphtheria vaccine is characterized in that, comprises the following steps:

[0023] 1) resuscitating, fermenting and culturing diphtheria bacteria strains to prepare diphtheria bacteria liquid;

[0024] 2) centrifuging the diphtheria bacterium liquid, filtering it three times, then performing ultrafiltration concentration, dialysis and washing, thereby obtaining diphtheria toxin;

[0025] 3) Detoxify the diphtheria toxin, and then use a tangential flow ultrafiltration system to perform ultrafiltration concentration, dialysis and washing;

[0026] 4) Purifying the detoxified diphtheria toxin.

[0027] As a preferred technical solution, the specific method of step 2) is: filter the diphtheria bacteria solution after centrifugation through a 0.22 μm → 0.1 μm filter membrane, then filter through a 300KD hollow fiber column, and then use a 10KD hollow fiber column to perform Concentrate by ultrafiltration, and when the...

specific Embodiment

[0032] Experiment 1) Diphtheria bacteria fermentation culture

[0033] Experimental Materials:

[0034] Strains: Bacillus diphtheriae PW8 strain (CMCC38007)

[0035] Medium: Improved synthetic medium: acid hydrolyzed casein 20g / L, peptone 5g / L, maltose 2.5g / L, lactic acid 2.5ml / L, Mueller's No. I solution 1ml / L, II solution 2ml / L ; The iron ion concentration of the culture medium is lower than 0.10mg / ml.

[0036] Rehydration: 20% maltose, Mueller's I, II solution, 1% glutamic acid solution.

[0037] Main equipment: 50L fermentation tank (Shanghai Wanmuqing Biological Engineering Co., Ltd.)

[0038] experimental method:

[0039] After the recovery of diphtheria strains, cultivate them on the improved liquid medium at 37°C for 72 hours, then scrape the bacterial film and spread it on the liquid medium to shake the flask, and cultivate at 37°C for 48 hours. Prepare 30L of liquid culture medium according to 20g / L of acid hydrolyzed casein, 5g / L of peptone, 2.5g / L of maltose, ...

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Abstract

The invention provides a preparation method of a diphtheria vaccine and aims at solving the problems of long production period of the diphtheria vaccine, ammonium sulfate pollution generated in production process, excessive impurity protein in the final product and over-large side reaction of the product in the prior art. The preparation method of the diphtheria vaccine is characterized by comprising the following steps of 1) carrying out resuscitation and fermentation cultivation of a corynebacterium diphtheriae strain, thereby preparing a diphtheria bacterial solution; 2) centrifuging the diphtheria bacterial solution, carrying out triple filtration treatment, and then carrying out ultrafiltration concentration, and dialysis and elution, thereby obtaining a diphtheria toxin; 3) detoxifying the diphtheria toxin, and then carrying out ultrafiltration concentration, and dialysis and elution by use of a tangential flow ultrafiltration system; 4) purifying the detoxified diphtheria toxin. High-speed centrifuging is used for removing bacteria, and ultrafiltration concentration and elution are used instead of adsorption by use of active carbon and salting out by use of ammonium sulfate, and the whole experimental steps can be finished by only one day, and therefore, the process steps are simplified, the production period is shortened, the production efficiency is improved and the controllability of the operation steps is improved.

Description

technical field [0001] The invention relates to the field of vaccines, in particular to a preparation method of a diphtheria vaccine. Background technique [0002] The diphtheria vaccine is made from diphtheria bacillus. The toxin secreted by the diphtheria bacillus is a single polypeptide chain without sugar groups and prosthetic groups. The protein is composed of 560 amino acids and has a molecular weight of 62kDa. Under the action of protease, it can be cut into notch toxin to have biological activity. Diphtheria toxoid includes two fragments, A and B, among which the B fragment is a hand-held binding and membrane-penetrating fragment, and the A fragment is a toxic fragment, which finally causes cell death by inhibiting the enzyme activity in protein synthesis. Toxicity can also occur, especially to the heart and peripheral and central nervous systems. [0003] The brief production process of the current diphtheria vaccine in my country is as follows: the strain is resu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/05C12P21/02C07K14/34C07K1/34A61P31/04C12R1/15
Inventor 耿星涛樊可陶亮马世勇许小媛贾福利刘彪贾涛
Owner SHANDONG YIDU BIOTECH
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