Large-scale preparation method for foot-and-mouth disease totivirus marked vaccine with high yield, high purity and high safety and product thereof

A large-scale preparation of foot-and-mouth disease virus technology, applied in the field of medicine and biology, can solve the problems of low content of effective antigen 146S particles, high total protein content, high impurity content, etc., to improve the yield of complete virus, high purity, and low side reaction rate Effect

Active Publication Date: 2015-04-08
吕宏亮 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the immune effect of the vaccine meets the requirements of the vaccine regulations, according to the safety requirements of modern vaccines, the content of impurities and total protein is high, and the content of effective antigen 146S particles is low. 21 Fine residual protein, bovine serum albumin, inactivators, preservatives, and chemical additives in the production process are not clearly defined and quantified. Although new vaccine quality standards were implemented in 2013, the quality standards and processes of vaccines should be continuously improved. To ensure that the vaccine is stable, safe and effective

Method used

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  • Large-scale preparation method for foot-and-mouth disease totivirus marked vaccine with high yield, high purity and high safety and product thereof
  • Large-scale preparation method for foot-and-mouth disease totivirus marked vaccine with high yield, high purity and high safety and product thereof
  • Large-scale preparation method for foot-and-mouth disease totivirus marked vaccine with high yield, high purity and high safety and product thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] The preparation of embodiment 1 high-yield, high-purity foot-and-mouth disease whole virus particle-labeled vaccine

[0076] Include the following steps:

[0077] a) BHK-21 cells are cultured in full suspension in a 100,000L bioreactor, and the cell density reaches 3-5×10 6 Inoculate foot-and-mouth disease virus cell-adapted strain (porcine foot-and-mouth disease virus / Mya98-XJ-2010 strain, prepared by Inner Mongolia Biwei Antai Biotechnology Co., Ltd.) according to the multiplicity of infection MOI0.01-0.1 of the virus, and prepare the virus stock solution with a stirring speed not exceeding 40rpm, cultivated for 4 days to harvest the virus liquid, use a preparative low-speed continuous flow centrifuge to remove cell debris, and harvest the supernatant and sediment at the same time, and the sediment was lysed in the presence of 0.2% Triton-X-100. The infected cells and cell membrane fragments were lysed. After repeated freezing and thawing 3 times, ultrasonication 3 t...

Embodiment 2

[0097] Example 2. Effect of Nucleolysis Step

[0098] 100000L bioreactor foot-and-mouth disease virus culture fluid centrifugation precipitation is through freeze-thawing, ultrasonication, adds 0.1% Triton X-100 (Sigma company product) in the technological operation, merges supernatant liquid and precipitation treatment liquid, adds Benzonase (Merck KgaA, 50units / ml ) and MgCl 2 (2mM) for 1 hour. The precipitate was removed by continuous flow centrifugation. Depth filtration adopts 0.8 micron and 0.45 micron filters successively (products of Sartorius, Germany), concentrates 5 times with 0.05 micron hollow fiber column, uses 6 times of volume of buffer solution containing 1.0M NaCl / 50mM Tris, pH 7.5 and 4 times of volume of buffer solution containing 0.4M NaCl / 50mM Tris dialyzed solution, pH 7.5. Concentrated dialysate was loaded on Sepharose Q-XL (Amersham) column, foot-and-mouth disease virus solution was eluted and collected with a buffer containing 0.55M NaCl / 50mM Tris, ...

Embodiment 3

[0102] Example 3 Buffer Liquid Replacement or Ultrafiltration, Concentration, Dialysis

[0103] 100000L bioreactor culture fluid was centrifuged, freeze-thawed, ultrasonic, centrifuged, Benzonase nuclease (50units / mi) was digested for 1 hour, 0.1% TritonX-100 was used for 30 minutes, and 0.5 micron filter, Millistak DE 30 / 60 filter ( Millipore company purchase) clarification, the clarified liquid is diluted to contain 0.3M NaCl, 300kD filter (Biomax 300, Pellicon 2module, Millipore company product) is concentrated 10 times with the same volume of buffer solution containing (0.6M NaCl / 50mM HEPES, pH 7.5), uses 2 times volume of dialysate containing (0.3MNaCl / 50mM HEPES pH 7), 2 times volume of dialysate containing (0.6MNaCl / 50mM HEPES pH 7.5), 2 times volume of dialysate (1.0M NaCl / 50mM HEPES pH 7.5), 3 times Volumetric dialysate (0.3M NaCl / 50mM HEPES pH 7.5) was dialyzed separately, and the conductivity and protein content were measured. It was found that when the NaCl salt co...

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Abstract

The invention discloses a large-scale preparation method for foot-and-mouth disease totivirus marked vaccine with high yield, high purity and high safety and a product thereof. The method comprises the following steps: a)collecting a virus solution; b)performing deep filtration on a membrane, performing ultrafiltration and performing enzymolysis on nuclease; c)purifying through a strong anion exchange adsorption bed or an adsorption film; d)depositing by PEG, extracting by chloroform-isoamyl aleohl; e)inactivating; F)performing density gradient centrifugation on an inactivation liquid through cane sugar and purifying; g)performing ultrafiltration dialysis and aseptic filtration; and h)reserving a stock solution or emulsifying. The provided foot-and-mouth disease totivirus marked vaccine antigen is uniform and complete foot-and-mouth virus particle, The vaccine is injected into body, so animal infection and immunization can be completely distinguished, does not contain foot-and-mouth disease virus non-structural protein and other virus particle, and does not contain animal-based foreign protein, polypeptide and oligopeptides, animal latent anaphylactic reaction, carcinogenesis and latent risk such as mad cow disease for causing animal infectious diseases due to vaccine injection can be effectively reduced, and the vaccine has no influence on animal food safety and trade.

Description

technical field [0001] The invention relates to a method for preparing a foot-and-mouth disease whole virus particle-labeled vaccine and its product, in particular to a method for large-scale preparation of a high-yield, high-purity, high-safety foot-and-mouth disease whole virus particle-labeled vaccine and its product. technology field. Background technique [0002] Foot-and-mouth disease (FMD) is a severe contact infectious disease of cloven-hoofed animals caused by foot-and-mouth disease virus (FMDV), which mainly harms cattle, sheep, pigs, camels, etc., and the incidence rate is extremely high. High, the transmission speed is extremely fast, and the World Organization for Animal Health lists it as the first class A zoonotic disease. Although the mortality rate of the disease is not high (except for young animals), it can cause a decline in animal production performance, and culling animals requires a lot of manpower, material and financial resources, and it will also a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/135C12N7/00A61P31/14C12R1/93
Inventor 吕宏亮张澍
Owner 吕宏亮
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