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
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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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