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Preparation method and application of porcine delta coronavirus inactivated vaccine

A coronavirus and inactivated vaccine technology, applied in the field of preparation of pig delta coronavirus inactivated vaccine, can solve the problems of high labor demand, high production cost, difficult to enlarge, etc., and achieves good safety, low production cost and easy effect of operation

Pending Publication Date: 2022-07-29
浙江洪晟生物科技股份有限公司 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are currently no PDCoV vaccines on the market both at home and abroad, and most of the vaccines are produced by adherent cells, which are not easy to scale up, have complicated processes, many labor requirements, and high production costs

Method used

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  • Preparation method and application of porcine delta coronavirus inactivated vaccine
  • Preparation method and application of porcine delta coronavirus inactivated vaccine
  • Preparation method and application of porcine delta coronavirus inactivated vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Step-by-step scale-up culture of ST whole suspension cells

[0028] 1.1 ST full suspension cell recovery: Take out the ST full suspension cell cryovial from the liquid nitrogen tank, immediately put it in a 37°C water bath, and shake the cryovial gently to thaw the liquid as soon as possible. The cell suspension was centrifuged at 1000 r / min for 5 minutes. Discard the supernatant, use 20ml serum-free medium (CD ST 258, purchased from Gansu Jianshun Biotechnology Co., Ltd.) for each cryovial to suspend the cells in a 125ml shake flask, set at 37°C, 130r / min, containing 5% CO. 2 Shaker culture; daily cell counts ( figure 1 ), when the cell density reaches 4~6×10 6 When pcs / ml, the seeding density is 1×10 6 Numbers / ml were subcultured and expanded into 500ml shake flasks, about 100ml.

[0029] 1.2 Amplification of ST full suspension cells: place a 500ml shake flask at 37°C for 130r / min, containing 5% CO 2 Shaker culture; count cells every day, when the cell...

Embodiment 2

[0030] Example 2: Screening of porcine deltacoronavirus ST full suspension cell culture conditions

[0031] 2.1 To explore the density of inoculated cells, inoculated dose and cell density during inoculation

[0032] Test method: when the cells grow to 6 × 10 6 When cells / ml or more, adjust the cell density to 3 × 10 in serum-free medium containing trypsin 6 pcs / ml, 4×10 6 pcs / ml, 5.0×10 6cells / ml for three cell densities, and the final concentration of trypsin after adjustment was 40 μg / ml. According to MOI of 0.0005, 0.001, 0.005, access to pig delta coronavirus (from Jiangsu Academy of Agricultural Sciences, GenBank: KU665558.1), 37 ℃ 130r / min containing 5% CO 2 Shaker culture. Samples were taken every 6 hours for cell counting, and the cell density was 1 to 1.5 × 10. 6 pcs / ml, 0.5~1×10 6 pcs / ml and 0.1~0.5×10 6 Samples / ml were sampled for virus content determination and sterility test, and the best technological conditions for virus culture were screened.

[0033]...

Embodiment 3

[0044] Example 3: Preparation of pig deltacoronavirus inactivated vaccine

[0045] 3.1 Production of porcine delta coronavirus culture solution: according to the method of Example 1, the ST full suspension cells were gradually enlarged into a 200L bioreactor for fermentation culture, and the culture volume was 100L. When the cells grew to 6 × 10 6 When the number / ml or more, the pig delta coronavirus was inserted according to the optimal process determined in Example 2. Specifically: when the cell density is 6 × 10 6 1 / ml, supplement with 6L trypsin (original concentration is 1mg / ml), 44L serum-free medium, 19ml porcine delta coronavirus (original titer is 10 7.50 TCID 50 / ml), continue the fermentation culture, and control the parameters of the reactor as follows: dissolved oxygen 40-50%, pH 7.0-7.2, stirring 80-100 rpm; sampling every 6 hours for cell counting, when the cell density drops to 0.5-1.0 × 10 6 Cell cultures were harvested at cells / ml.

[0046] 3.2 Removal of...

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Abstract

The invention discloses a preparation method of a porcine delta coronavirus inactivated vaccine. The preparation method comprises the following steps: 1) step-by-step amplification culture of ST full suspension cells in a serum-free culture medium; 2) performing fermentation culture on the ST full suspension cells subjected to amplification culture, when the cells grow to 6 * 10 < 6 > / ml or above, diluting the cells to 4-5 * 10 < 6 > / ml by using a serum-free culture medium containing pancreatin, enabling the final concentration of the pancreatin in the serum-free culture medium to be 20-30mu g / ml at the moment, and inoculating the porcine delta coronavirus according to the MOI of 0.0005-0.001; 3) when the cell density is reduced to (0.5-1.0) * 10 < 6 > / ml, harvesting a cell culture; (4) removing cell debris from the harvested cell culture under a sterile condition and carrying out inactivation treatment; and 5) mixing and emulsifying the virus liquid passing the inactivation inspection and a pharmaceutically acceptable adjuvant according to a certain proportion, and quantitatively subpackaging to obtain the vaccine. The method disclosed by the invention is simple in process, low in production cost, low in labor consumption and high in virus titer.

Description

technical field [0001] The invention belongs to the field of biotechnology and preventive veterinary medicine, and specifically relates to a preparation method and application of a pig delta coronavirus inactivated vaccine. Background technique [0002] Porcine deltacoronavirus (PDCoV) belongs to the genus deltacoronavirus and is a newly discovered porcine coronavirus in recent years. It can cause severe diarrhea, dehydration and vomiting in pigs, especially suckling pigs. fatality rate. It has now spread all over the world, causing major economic losses in the pig industry and attracting global attention. However, at present, there is no PDCoV vaccine on the market at home and abroad, and most of the vaccines are produced by adherent cells, which are not easy to scale up, complicated in process, high labor demand and high production cost. Therefore, it is of great significance to develop a pig delta coronavirus vaccine with good immune effect, safety and reliability, easy...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/215A61K39/39A61P31/14C12N7/00C12N7/06C12R1/93
CPCA61K39/12A61K39/39A61P31/14C12N7/00A61K2039/552A61K2039/5252A61K2039/55511C12N2770/20051C12N2770/20063C12N2770/20034
Inventor 舒建洪李彬李肖梁查银河余晓玉何玉龙冯华朋李基棕张稳涛
Owner 浙江洪晟生物科技股份有限公司
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