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A kind of preparation method of foot-and-mouth disease purified vaccine
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A culture medium and cell technology, applied in the biological field, can solve the problems of large immune side effects, short immune period, and low antibody level, and achieve the effect of eliminating emergency response, ensuring safety, and ensuring safety
Inactive Publication Date: 2015-11-18
北京必威安泰科技有限公司 +1
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[0004] There are two main problems in the existing inactivated foot-and-mouth disease vaccines: 1. The immune protection is not stable, mainly manifested in: after immunization, neutralizing antibodies cannot be detected or the antibody level is very low, or the immunization period is short, only 2 to 3 2. Large immune side effects, mainly: the downstream production process is still the traditional method without purification treatment, there are some impurities and allergic substances in the culture medium and host cells in the vaccine, and the effective antigen content is low
This has a great impact on the prevention and control of foot-and-mouth disease
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Embodiment 1
[0028] The production of embodiment 1 foot-and-mouth disease virus liquid
[0029] 1. Culture of BHK-21 cells
[0030] Prepare serum-free or animal-derived component-free medium (1 L) according to the following recipe:
[0031] 380mgL-Alanine, 90mgL-Arginine Hydrochloride, 280mgL-Aspartic Acid, 70mgL-Cysteine Hydrochloride, 120mgL-Glutamic Acid, 50mgL-Arginine, 100mgL-Tyrosine, 130mgL-Valine Acid, 2.5g sucrose, 100mg sodium citrate, 7.5gNaCl, 300mgKCl, 200mgCaCl 2 And 20mg of phenol red, distilled water to make up 1000ml.
[0032] Serum-free Microcarrier Suspension Culture of BHK-21 Cells BHK-21 cells are from ATCC, the working cell bank cell passage should not exceed 10 passages of the main cell bank, and the BHK-21 working cells should be within 10 passages. Serum-free culture medium of the present invention is added Cytodex-3 microcarrier according to 5~8g / ml, cultivates 6-7 days with 1.5L bioreactor 37 ℃, stirring speed 20rpm, carries out digestion again, transfers to...
Embodiment 2
[0043] The inactivation of embodiment 2 purifying foot-and-mouth disease virus
[0044] The purified virus solution and the crude virus solution prepared in Example 1 were sterilized and filtered with a 0.22 μm membrane and added with 1 / 4000 β propiolactone to inactivate at 37°C for 3-4 days, and stored at 4°C for use.
Embodiment 3
[0045] Example 3 Vaccine Preparation
[0046] The inactivated purified inactivated solution and crude virus solution were diluted with 199 medium (pH 7.2) and subpackaged to contain 20 μg of each serotype antigen per head. See Table 2 for the comparison between the prepared purified vaccine and the crude vaccine.
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Abstract
The invention discloses a method for preparing a purified foot-and-mouth disease vaccine, a serum or animal-derived ingredient free culture medium and an application of the serum or animal-derived ingredient free culture medium to the preparation of the foot-and-mouth disease vaccine, belonging to the filed of biotechnology. The method for preparing the purified foot-and-mouth disease vaccine comprises the following steps of: culturing a foot-and-mouth disease virus by using the serum or animal-derived ingredient free culture medium, purifying an obtained virus solution to obtain a purified antigen, subjecting a cell strain BHK-21 or BSR to the multiple-generation acclimatization culture and the suspension culture by 300L of a microcarrier through the serum-free culture medium, inoculating the cell strain BHK-21 or BSR against the foot-and-mouth disease vaccine, stirring at the rotating speed of 30-50rpm, microfiltrating, ultrafiltrating, concentrating 50-200 times, carrying out chromatography with a Sephawse6FF molecular sieve or density gradient zonal centrifugation with a continuous flow, and inactivating with beta-propiolactone to obtain the serotype univalent or multivalent vaccine for cattle, sheep and pigs.
Description
technical field [0001] The invention relates to a preparation method of a purified foot-and-mouth disease vaccine, and also relates to a serum-free or animal-derived component-free culture medium and its application in preparing the foot-and-mouth disease vaccine. Belongs to the field of biotechnology. Background technique [0002] Foot-and-mouth disease is an acute, highly contagious, febrile infectious disease of artiodactyls caused by Foot-and-mouth disease virus (FMDV), known for its rapid spread and high infection rate. Once the disease breaks out, it will cause huge economic losses to the affected country. Countries all over the world attach great importance to the research of this disease, and the International Veterinary Bureau ranks it as the first class A infectious disease. Foot-and-mouth disease is a member of the genus Foot-and-Mouth Virus in the family Picornaviridae, and has seven serotypes: A, O, C, AsiaⅠ, SAT1, SAT2 and SAT3. At present, the prevention and...
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