Optimized process method for amplifying enterovirus type 71 by use of bioreactor
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A bioreactor and enterovirus technology, applied in the biological field, achieves high repeatability and stability, saves manpower and material costs, and reduces the difficulty of purification
Active Publication Date: 2013-07-24
ZHEJIANG PUKANG BIOTECH
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Hydrolyzed milk protein is a safe and effective medium additive, which has been widely used. It has not been reported to be used in the a
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Embodiment 1
[0033] Bioreactor: China Hangzhou Anpu Bioengineering Co., Ltd. 10L torrent bioreactor with a working volume of 5L, model: AP20sc;
[0034] Microcarrier: Fibra disk paper carrier (Hangzhou Anpu Bioengineering Co., Ltd.);
[0035] Virus: enterovirus EV71 Zhejiang strain C4 subtype; inoculation MOI value is about 0.5;
[0036] Cell growth medium: DMEM medium containing 10% serum by volume (GIbco);
[0037] Cell maintenance solution: DMEM medium containing 0.5% hydrolyzed milk protein (Gibco);
[0038] Bioreactor cell culture: 150g of sterilized Fibra disk carrier has been added to the 10L reactor, soaked overnight in phosphate buffer solution PBS with pH 7.2, discarded and added to cell growth medium, and inoculated with VERO cells, the inoculation amount is ( 2.2±0.3)×10 9 cells to be cultured. The culture parameters were set as follows: pH 7.2-7.6, temperature 37°C, dissolved oxygen 50-80%, stirring speed 50-60 revolutions per minute (rpm). Samples were taken regularly ev...
Embodiment 2
[0045] Bioreactor: NBS 5L cell reactor, model: Bioflo310 (NBS Company, USA), working volume is 4L;
[0046] Microcarrier: Fibra disk paper carrier (NBS Company, USA);
[0047] Virus: Enterovirus 71 (EV71) subtype C4 (Zhejiang strain); MOI of inoculation is about 0.5;
[0048] Cell growth medium: DMEM medium containing 10% serum by volume (GIBCO);
[0049] Cell maintenance solution: DMEM medium containing 0.5% hydrolyzed milk protein (GIBCO company);
[0050] Cell culture: 150g of sterilized Fibra disk carrier has been added to the 5L reactor, soaked overnight in phosphate buffered saline solution PBS of pH 7.2, after discarding, add 4L of cell growth medium as a working volume, inoculate VERO cells, inoculum volume 2.3×10 9 cells to be cultured. The culture parameters were set as: pH 7.2-7.6, temperature 37°C, dissolved oxygen 50-80%, stirring speed 50rpm. Samples were taken regularly every day to measure glucose consumption, so as to estimate cell growth. Under stable co...
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Abstract
The invention relates to the field of biotechnology, and in particular relates to an optimized process method for amplifying the enterovirus type 71. The method adopts a poly-fiber paper scrap as a carrier and amplifies an African green monkey kidney cell by use of a bioreactor so as to establish a whole process flow for copying and amplifying the enterovirus type 71. Under a condition of completely adapting to an enterovirus type 71 copying and amplifying system, the method adopts a DMEM (dulbecco's minimum essential medium) containing 10% of serum in a cell culture stage; after virus inoculation and adsorption for 24 hours, the method adopts a DMEM containing 3-5% of serum; 0.5% of lactoalbumin hydrolysate is added by use of a serum-free medium in a virus harvesting stage, and 2g/L of glucose is supplemented every 24 hours; and on the basis of reducing the later-period purifying difficulty and adapting to the requirements for a biological product, the optimized process realizes a higher virus titer. The method provided by the invention has good repeatability and efficient enterovirus type 71 amplifying process, and can be used for amplifying the enterovirus type 71 by use of a bioreactor by taking a poly-fiber paper scrap as a carrier.
Description
technical field [0001] The invention relates to the field of biotechnology, in particular to a set of comprehensively optimized process methods for amplifying enterovirus type 71 (EV71). Background technique [0002] Enterovirus 71 (EV71) is one of the important pathogens of hand, foot and mouth disease. The complications of hand, foot and mouth disease caused by EV71 infection are relatively serious, and the mortality rate is relatively high. Research hotspots. At present, the large-scale culture technology of green monkey kidney (Vero) cells is relatively mature, but there are few reports on the culture of EV71 virus. At present, some progress has been made in culturing EV71 with Cytodex1 as a carrier. ) carrier has not been reported yet. [0003] To perfect this process, it must first be based on a comprehensive understanding of the physiological and growth characteristics of VERO cells, as well as a comprehensive grasp of the replication mechanism of EV71-infected cell...
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